| 1. |
- Outchkourov, NS, et al.
(författare)
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Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Franklinielia occidentalis) in greenhouse trials
- 2004
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7652 .- 1467-7644. ; 2:5, s. 449-458
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Tidskriftsartikel (refereegranskat)abstract
- Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripiclae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom-made, multiclomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases [kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin (EIM)] were fused into reading frames consisting of four (K-A-C-P) to five (EIM-K-A-C-P) proteins, and were shown to fold into functional inhibitors in the yeast Pichia pastoris. The multiclomain proteins were expressed in potato and found to be more resistant to degradation by plant proteases than the individual domains. In a time span of 14-16 days, transgenic potato plants expressing EIMKACP and KACP at a similar concentration reduced the number of larvae and adults to less than 20% of the control. Leaf damage on protected plants was minimal. Engineered multiclomain cysteine protease inhibitors thus provide a novel way of controlling western flower thrips in greenhouse and field crops, and open up possibilities for novel insect resistance applications in transgenic crops.
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| 2. |
- Hofvander, P, et al.
(författare)
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Field performance and starch characteristics of high-amylose potatoes obtained by antisense gene targeting of two branching enzymes
- 2004
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7652 .- 1467-7644. ; 2:4, s. 311-320
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Tidskriftsartikel (refereegranskat)abstract
- Potato is an important crop for starch production, but there are limitations regarding the genetic variation of starch quality. in maize, starches with various properties have been available for a long time by mutational breeding. Amylose starch from potatoes differs from cereal amyloses in several functionally important aspects, such as a higher degree of polymerization. Areas of application in which the degree of polymerization is of importance include film forming and the polymeric properties of bioplastics. High-amylose potato lines have been achieved by inhibiting the two known branching enzyme forms of potato. A single inserted gene construct for the inhibition of both forms resulted in structural changes of the starch to levels of branching that were below the commercially available amylose standards of potato. The high-amylose potato lines were tested in multiple year field trials of agronomic performance and were used for the pilot plant production of starch. The introduced trait was confirmed to be stable over multiple years. The consequences of the modification were found to be an increased tuber yield, reduced starch content, smaller granule size and an increase in reducing sugars.
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| 3. |
- Outchkourov, NS, et al.
(författare)
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Specific cysteine protease inhibitors act as deterrents of western flower thrips, Frankliniella occidentalis (Pergande), in transgenic potato
- 2004
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7652 .- 1467-7644. ; 2:5, s. 439-448
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed.
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| 4. |
- Gutierrez, Laurent, et al.
(författare)
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The lack of a systematic validation of reference genes : a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.
- 2008
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.
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| 5. |
- Tsai, Alex Y-L, et al.
(författare)
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Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure
- 2012
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Ingår i: Plant Biotechnology Journal. - : John Wiley & Sons. - 1467-7644 .- 1467-7652. ; 10:9, s. 1077-1087
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Tidskriftsartikel (refereegranskat)abstract
- A family 15 carbohydrate esterase (CE15) from the white-rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col-0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl-4-O-methyl-d-glucopyranuronate and could target ester linkages that contribute to lignin-carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf-yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT-IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross-linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross-linking within plant cell walls.
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| 6. |
- Wiklund, Susanne, et al.
(författare)
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A new metabonomic strategy for analysing the growth process of the poplar tree
- 2005
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 3:3, s. 353-362
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Tidskriftsartikel (refereegranskat)abstract
- High-resolution, magic angle spinning, proton nuclear magnetic resonance (H-1 HR/MAS NMR) spectroscopy and multivariate data analysis using batch processing (BP) were applied to the analysis of two different genotypes of poplar tree (Populus tremula L. x tremuloides Michx.) containing an antisense construct of PttMYB76 and control (wild-type). A gene encoding a MYB transcription factor, with unknown function, PttMYB76, was selected from a cambial expressed sequence tag (EST) library of poplar tree (Populus tremula L. x tremuloides Michx.) for metabonomic characterization. The PttMYB76 gene is believed to affect different paths in the phenyl propanoid synthetic pathway. This pathway leads to the formation of S- and G-lignin, flavonoids and sinapate esters. Milled poplar samples collected at the internodes of the tree were analysed using H-1 HR/MAS NMR spectroscopy. The application of multivariate BP of the NMR results revealed a growth-related gradient in the plant internode direction, as well as the discrimination between the trees with down-regulated PttMYB76 expression and wild-type populations. This paper focuses on the potential of a new analytical multivariate approach for analysing time-related plant metabonomic data. The techniques used could, with the aid of suitable model compounds, be of high relevance to the detection and understanding of the different lignification processes within the two types of poplar tree. Additionally, the findings highlight the importance of applying robust and organized multivariate data analysis approaches to facilitate the modelling and interpretation of complex biological data sets.
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| 7. |
- Gray-Mitsumune, Madoka, et al.
(författare)
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Ectopic expression of a wood-abundant expansin PttEXPA1 promotes cell expansion in primary and secondary tissues in aspen
- 2008
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 6:1, s. 62-72
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Tidskriftsartikel (refereegranskat)abstract
- Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an alpha-expansin PttEXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen (Populus tremula L. x P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. PttEXPA1 belongs to Subfamily A of alpha-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose-xyloglucan composites in vitro, indicating that PttEXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that PttEXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta. Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. PttEXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition.
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| 8. |
- Li, Xue-Yuan, et al.
(författare)
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Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica
- 2012
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 10, s. 862-870
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Tidskriftsartikel (refereegranskat)abstract
- Erucic acid (22 : 1) is a major feedstock for the oleochemical industry. In this study, a gene stacking strategy was employed to develop transgenic Crambe abyssinica lines with increased 22 : 1 levels. Through integration of the LdLPAAT, BnFAE1 and CaFAD2-RNAi genes into the crambe genome, confirmed by Southern blot and qRT-PCR, the average levels of 18 : 1, 18 : 2 and 18 : 3 were markedly decreased and that of 22 : 1 was increased from 60% in the wild type to 73% in the best transgenic line of T4 generation. In single seeds of the same line, the 22 : 1 level could reach 76.9%, an increase of 28.0% over the wild type. The trierucin amount was positively correlated to 22 : 1 in the transgenic lines. Unlike high erucic rapeseed, the wild-type crambe contains 22 : 1 in the seed phosphatidylcholine and in the sn-2 position of triacylglycerols (5% and 8%, respectively). The transgenic line with high 22 : 1 had decreased 22 : 1 level in phosphatidylcholine, and this was negatively correlated with the 22 : 1 level at the sn-2 position of TAG. The significances of this study include (i) achieving an unprecedented level of 22 : 1 in an oil crop; (ii) disclosing mechanisms in the channelling of a triacylglycerol-specific unusual fatty acid in oil seeds; (iii) indicating potential limiting factors involved in the erucic acid biosynthesis and paving the way for further increase of this acid and (iv) development of an added value genetically modified oil crop having no risk of gene flow into feed and food crops.
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| 9. |
- Ståhl, Ulf, et al.
(författare)
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A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine
- 2009
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 7, s. 602-610
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Tidskriftsartikel (refereegranskat)abstract
- P>The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cis delta 9,12) and 18:3(cis delta 9,12,15)) at the expense of very long monounsaturated FA (20:1(cis delta 11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from delta YOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.
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| 10. |
- Abrouk, Michael, et al.
(författare)
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The in silico identification and characterization of a bread wheat/Triticum militinae introgression line : Characterization of alien introgression in wheat
- 2017
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Ingår i: Plant Biotechnology Journal. - : John Wiley & Sons. - 1467-7644 .- 1467-7652. ; 15:2, s. 249-256
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Tidskriftsartikel (refereegranskat)abstract
- The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross bread wheat cv. Tähti ⨯ T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed RICh (rearrangement identification and characterization) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2,132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbors 131 homoeologs out of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should facilitate not only gene isolation, but may also be informative with respect to chromatin structure and behavior studies.
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