Plant Biotechnology Journal

Cover image for Vol. 13 Issue 2

Edited By: Henry Daniell

Impact Factor: 5.677

ISI Journal Citation Reports © Ranking: 2013: 13/199 (Plant Sciences); 16/165 (Biotechnology & Applied Microbiology)

Online ISSN: 1467-7652

Featured Articles

Vol. 10 Iss. 4

Transposon tagging in diploid strawberry

Veilleux et al.

Transposon tagging exploits the ability of maize transposable elements to move from one genomic site to another, in the process, changing gene expression of nearby genes. It is an essential tool for forward and reverse genetics research, providing a means to study gene function either by interrupting native genes or by overexpressing them. The development of hundreds of thousands of transposon tagged lines in Arabidopsis and rice has depended upon transformation with vectors harboring the maize elements, followed by their expression and transposition in an alien system. The study of altered gene expression in these lines may reveal a native gene’s function, thus enabling new or revised gene descriptions. However, the extensive collections of transposon tagged lines in Arabidopsis and rice do not encompass any gene models for the development of fleshy fruit, a process that is basic to the production of many horticultural crops. To fill this gap, we set about to test a transposon tagging system in the emerging model fruit crop, Fragaria vesca. The essential functional components include: expression of maize transposase in an alien system, excision and re-integration of the maize dissociator (Ds) element in the alien genome, and a facile system to determine the sites of transposition. Here we report the development of the first available transposon tagged lines for diploid strawberry. We made extensive use of the recently available strawberry genome sequence to position our tagged lines along the linkage groups and found global transposition of Ds from the original transformation site to all of the other linkage groups. This study provides groundwork for future efforts to generate unlimited numbers of transposon tagged lines in strawberry that will theoretically tag any gene of interest to discover novel gene functions in fruit crops.

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Vol. 10 Iss. 4

Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification
Aymerick Eudeset al. 

Plant cell walls are source of the most abundant organic materials on Earth and are mainly composed of cellulose, hemicellulose and lignin. This lignocellulosic biomass represents a large resource of fermentable sugars for the microbial production of chemicals such as biofuels, provides feeds for ruminants, and is the source of cellulose for pulp and paper production. Lignin confers recalcitrance to this biomass and impedes the extraction and/or hydrolysis into simple sugars of the polysaccharide polymers. The work presented by Eudes et al. describes a strategy for the in-planta overproduction of rare lignin monomers synthesized upon expression of a bacterial enzyme in tissues developing lignified cell walls. This enzyme removes the propanoid tail of monolignol precursors, and lignins produced in these engineered plants incorporate higher amount of the generated truncated-side chain monomers that causes a decreased molecular weight of lignin polymers. Consequently, cell walls from these plants are less recalcitrant during enzymatic saccharification treatments leading to improved sugar releases. Importantly, specific expression of the bacterial enzyme to the lignifying tissues rich in secondary cell walls did not affect plant development, nor did reduce the amount of lignin which is essential for plant growth. This pilot study conducted in a plant model organism provides some knowledge towards further design of biofuel crops with both reduced cell wall recalcitrance and uncompromised biomass yield.
We also predict that reduction of lignin molecular weight should enhance its extractability and facilitate cellulose purification during pulping procedures, and also expect that this lignin engineering strategy is applicable to forage crops to improve their digestibility.

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Vol. 10 Iss. 4

Enhancing ascorbate in fruits and tubers through over-expression of the l-galactose pathway gene GDP-l-galactose phosphorylase
Sean Bulley et al.

Ascorbate or vitamin C is necessary for humans as our primate ancestors lost the ability to synthesise ascorbate because of a mutation in the gene encoding the enzyme for the final step in the ascorbate pathway. Consequently, humans obtain most of their vitamin C from plant sources. Current recommendations for vitamin C intake for humans are in the range of 40–90 mg per day and even higher levels of ascorbate may be beneficial. Many food sources of vitamin C are relatively low in vitamin C, so it can be difficult to achieve high intakes without synthetic supplements. Consequently, there is a significant opportunity to improve human vitamin C intake by developing higher vitamin C plant-based foods.

The main pathway to ascorbate in many plants is the L-galactose pathway. We have previously shown that stably transforming the gene for GDP-galactose phosphorylase (GGP, also known as VTC2), into Arabidopsis increased leaf ascorbate up to threefold and had similar effects in transiently transformed tobacco leaves. These effects are among the largest noted for any gene in the L-galactose pathway. In this work we transformed three important commercial crops (potato, strawberry and tomato) with GGP to investigate the potential for and effects of increased vitamin C content in the tubers and fruits of these plants.

In potato we increased tuber vitamin C from about 10 to over 36 mg/100g FW (fresh weight) without affecting yield or appearance. In tomato we increased ascorbate from less than 20 to up to over 110 mg/100g, although as fruit vitamin C increased the fruit tended to be smaller and seed production aborted early. The fruit shape changed, becoming fasciated and the fruit had no mucilage typically surrounding the seed. These fruit may be excellent for tomato paste production as they contained less water, if propagation using clonal methods was commercially acceptable or the gene was turned off in seed production lines. Strawberry fruit already have a high vitamin C level (around 60 mg/100g FW) but we still managed to raise fruit vitamin C two fold, again without affecting fruit appearance.

Calculations based on vitamin C content of these biotechnologically produced nutritionally enhanced fruit with increased natural vitamin C suggested that daily vitamin C supply from transgenic potatoes could be met with 1/3 the fresh weight of regular tuber, while transgenic tomato and strawberry fruit would provide more than the daily recommended amount with less than 100g of fruit.

This information can also be used to further the breeding crops with high vitamin C through marker selected selection using gene markers for GGP.

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Vol. 10 Iss. 2 Overexpression of GCN2-type protein kinase in wheat has profound effects on free amino acid concentration and gene expression
Edward H. Byrne, Ian Prosser, Nira Muttucumaru, Tanya Y. Curtis, Astrid Wingler, Stephen Powers and Nigel G. Halford

Interest in free amino acids in grains, tubers and other crop products has been stimulated in recent years because of their participation in the Maillard reaction. This complex series of non-enzymic reactions between amino groups and reducing sugars during high-temperature cooking or processing gives rise to a plethora of compounds that impart flavour, aroma and colour, but also produces undesirable contaminants such as acrylamide and furans. Acrylamide forms when the amino acid that participates in the final stages of the reaction is asparagine. In this issue, Byrne et al. describe the over-expression of a general control nonderepressible-2 (GCN2)-type protein kinase, TaGCN2, in transgenic wheat, with profound effects on free amino acids, notably a more than two thirds reduction in free asparagine concentration. GCN2-type protein kinases are present in all eukaryotes and phosphorylate the alpha subunit of translation initiation factor 2 (eIF2α), thereby regulating protein synthesis in response to amino acid availability and a variety of stresses. In yeast, GCN2 also controls amino acid synthesis through the action of a transcription factor, GCN4. Byrne et al. showed that TaGCN2 over-expression in wheat affected the expression of a suite of genes encoding enzymes involved in amino acid synthesis, including asparagine synthetase. They went on to show that sulphur deficiency-induced activation of asparagine synthetase gene expression occurred in wild-type plants but not in TaGCN2 over-expressing lines, unexpectedly implicating TaGCN2 in sulphur signalling. The study suggests that manipulation of TaGCN2 gene expression could be used to reduce free asparagine accumulation in wheat grain and the risk of acrylamide formation in wheat products. It demonstrates a role for TaGCN2 in regulating gene expression and is the first to implicate GCN2-type protein kinases so clearly in sulphur signalling in any organism.

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Vol. 10 Iss. 2

Cisgenic barley with improved phytase activity
Inger B. Holme, Giuseppe Dionisio, Henrik Brinch-Pedersen, Toni Wendt, Claus K. Madsen, Eva Vincze and Preben B. Holm

"Cisgenic barley with improved phytase activity" describes a cisgenic approach for improving the bioavailability of phytate-bound phosphate in the barley grain. The natural allelic variation for phytase activity is limited in barley and classical breeding is therefore difficult. Numerous transgenic crop species with improved phytase activity derived from microbial phytases have been developed but commercialization has lacked behind. Cisgenesis implies that the plant is transformed only with its own or very closely related genetic material and all foreign gene sequences should be absent in the final cisgenic plant. Surveys in Europe have shown that cisgenic crops are more acceptable to the public than transgenic crops and cisgenic crops may be subjected to less rigid regulatory measures in the future. In this study, cisgenic barley with a 2.8 fold increase in phytase activity was generated by inserting a single extra copy of the genomic barley phytase gene, HvPAPhy_a, expressed during grain filling. The study illustrates that genetic modification according to the cisgenesis concept is technically possible in barley. Insertion of extra copies of endogenous genes might be a useful and publicly acceptable tool for the improvement of traits with limited natural allelic variation.

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Vol. 10 Iss. 1

WRKY transcription factors: key components in abscisic acid signalling
Deena L. Rushton, Prateek Tripathi, Roel C. Rabara, Jun Lin, Patricia Ringler, Ashley K. Boken, Tanner J. Langum, Lucas Smidt, Darius D. Boomsma, Nicholas J. Emme, Xianfeng Chen, John J. Finer, Qingxi J. Shen, Paul J. Rushton 

For over fifteen years, most research concerning the WRKY family of transcription factors has centered on their roles in regulating plant responses to biotic stress, notably as regulators of the plant immune response. More recently, it has become clear that WRKY proteins are also involved in regulating responses to abiotic stress. However, the mechanisms involved and the positions of WRKY proteins in abiotic stress-inducible signalling networks have remained unclear. Recent significant progress in ABA research has now placed specific WRKY proteins firmly in ABA-responsive signalling pathways, where they act downstream of at least two ABA receptors: the cytoplasmic PYR⁄PYL⁄RCAR-protein phosphatase 2C-ABA complex and the chloroplast envelope–located ABAR–ABA complex. Members of the WRKY family are also found both upstream and downstream of well-characterized ABA-responsive genes such as ABI5. These new observations show that WRKY transcription factors are key nodes in ABA-responsive signalling networks.

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Vol. 9 Iss. 9

Attitudes of European farmers towards GM crop adoption
Francisco J. Areal, Laura Riesgo, Emilio Rodríguez-Cerezo

This article analyses European Union (EU) farmers’ attitudes towards adoption of genetically modified crops by identifying and classifying groups of farmers. Cluster analysis provided two groups of farmers allowing us to classify farmers into potential adopters or rejecters of genetically modified herbicide-tolerant (GMHT) crops. Results showed that economic issues such as the guarantee of a higher income and the reduction of weed control costs are the most encouraging reasons for potential adopters and rejecters of GMHT crops. This article also examines how putting in place measures to ensure coexistence between GM and non-GM crops may influence farmers’ attitudes towards GMHT crop adoption. Results show that the implementation of a coexistence policy would have a negative impact on farmers’ attitudes on adoption and consequently may hamper GMHT adoption in the EU.

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Vol. 9 Iss. 8

Transgenic Pm3b wheat lines show resistance to powdery mildew in the field
Brunner et al

The work presented by Brunner et al. is a model study on the transgenic use of a resistance (R) gene in the crop plant wheat (Triticum aestivum L.). The wheat lines analysed overexpress the Pm3b resistance gene and were tested in different field environments. They showed increased resistance against wheat powdery mildew, a significant fungal pathogen, during several field seasons. Many R genes are present in wild relatives of crop plants and are only of limited use in classical breeding because of the difficulty of introgression. However, they might be used as transgenes in future breeding work. Therefore, this study reports on a promising strategy for improvement of disease resistance. Since a total of 17 functional alleles of the used resistance locus Pm3 were cloned previously, this work represents a first step in the production of multilines which are predicted to prolong the durability of the used Pm3 alleles. Finally, the work describes field trials which were the basis for several research projects in a large consortium on benefits and risks (of the deliberate release) of genetically modified plants.

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