Lignin Content In Transgenic Plants Research Articles

Ectopic expression of an antisense BpCCoAOMT gene from Betula platyphylla Suk. affects growth and development of tobacco due to lignin content reduction

Lignin is an aromatic polymer and macromolecular organic material and is one of the major components of plant cellular walls. It provides mechanical support for plants and protection against pathogen invasion. Caffeoyl-CoA O-methyltransferase (CCoAOMT) is recognized to be involved in phenylpropanoid metabolism and lignin synthesis, and plays an important role in precursor synthesis of G-lignin units. In this study, a gene encoding CCoAOMT (Genbank ID: AY860952) was isolated from birch (Betula platyphylla Suk.). The predicted BpCCoAOMT polypeptide had high affinity with CCoAOMT from other species. Real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that BpCCoAOMT is most highly expressed in young stems. To study its function in vivo, antisense BpCCoAOMT complementary deoxyribonucleic acid (cDNA) was transformed into tobacco (SR-1) by the Agrobacterium tumefaciens-mediated method. The expression of NtCCoOAMT in antisense BpCCoAOMT transgenic tobacco was down-regulated and total lignin content of transgenic plants decreased by 39% compared to control plants. Maule reagent was used to distinguish lilac lignin and guaiac wood lignin in situ, which enables S-lignin to exhibit a specific red response. Results revealed that all transgenic tobacco plants were dark brown, while controls were dark red. This indicated that S-lignin content in the xylem was reduced. Compared to wild-type (WT) plants, transgenic tobacco plants had delayed flowering and some had slender stems, curling, and easy lodging. This indicates that the decrease in lignin content interferes with the normal growth of plants.

Elicitors and defense gene induction in plants with altered lignin compositions.

A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositions led to differential extractability of a range of heterogeneous oligosaccharide epitopes, with elicitor activity originating from different cell wall polymers. Alteration of lignin composition affects interactions with plant cell wall matrix polysaccharides to alter the sequestration of multiple latent defense signal molecules with an impact on biotic stress responses.

Establishment of Miscanthus sinensis with decreased lignin biosynthesis by Agrobacterium–mediated transformation using antisense COMT gene

This study was to determine a transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of an antisense caffeic acid O-methyltransferase gene in the M. sinensis (MsCOMT-AS). Transformation of callus derived from seeds and immature inflorescences of M. sinensis was established by using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish the stable transformation system, several transformation factors such as explant type, strain, co-culture periods, acetosyringone concentration, and selective markers were assessed. In this study, seven putative transgenic plants were obtained from callus transformation and plantlet regeneration. Various tests including PCR analysis and RT-PCR were used to detect the transgenic insert. The transgenic plants were also characterized for their agronomic and morphological characteristics, expression of MsCOMT-AS gene, and variation in lignocellulosic content. Biomass related traits such as plant height, number of leaves, length of leaf, stem diameter, fresh weight, dry weight, and cell size of the control plants were superior to transgenic plants. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose content in transgenic plants were not increased. Variation in cellulose and hemicellulose content had no correlation with variation in lignin content of transgenic plants. In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance

BackgroundThe mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential.ResultsA BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth.ConclusionsA TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

Genetic Modification of Lignin Biosynthetic Pathway in Populus ciliata Wall. via Agrobacterium-Mediated Antisense CAD Gene Transfer for Quality Paper Production

The present studies describe the development of transgenic Himalayan poplar (Populus ciliata Wall.) plants via Agrobacterium-mediated antisense cinnamyl alcohol dehydrogenase (CAD) gene transfer for genetic modification of lignin biosynthetic pathway for lowering down the lignin content by post-transcriptional silencing of CAD gene. Agrobacterium tumefaciens strain C58C1rifR(pMP90) harboring CAD gene in antisense orientation and npt-II gene in a binary vector p35SASCAD was used for genetic transformation experiments. By combining the best treatments of 48 h pre-culturing and 48 h co-cultivation time periods, a transformation frequency of 14 % was obtained from petiole explant of P. ciliata. Out of total 9 randomly selected putative transgenic plants of P. ciliata, five were found to show the presence and integration of npt-II gene in poplar genome as confirmed by PCR using npt-II gene specific primers. npt-II gene which is transferred along with antisense CAD gene (in the same gene construct), got amplified with gene specific primers at 0.7 Kb. The lignin content of transgenic plants of P.ciliata was found to be reduced by a fraction of 3–4 % as compared to the control plants after 4 months of transfer to the greenhouse conditions.