Transgenic miR156 Switchgrass in the Field: Growth, Recalcitrance and Rust Susceptibility

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. Highexpressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development

Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance

Tissue-specific expression of Populus C \u3c inf\u3e 19 GA 2-oxidases differentially regulate above- and below-ground biomass growth through control of bioactive GA concentrations

Summary: • Here, we studied the poplar C 19 gibberellin 2-oxidase (GA2ox) gene subfamily. We show that a set of paralogous gene pairs differentially regulate shoot and root development. • PtGA2ox4 and its paralogous gene PtGA2ox5 are primarily expressed in aerial organs, and overexpression of PtGA2ox5 produced a strong dwarfing phenotype characteristic of GA deficiency. Suppression of PtGA2ox4 and PtGA2ox5 led to increased biomass growth, but had no effect on root development. By contrast, the PtGA2ox2 and PtGA2ox7 paralogous pair was predominantly expressed in roots, and when these two genes were RNAi-suppressed it led to a decrease of root biomass. • The morphological changes in the transgenic plants were underpinned by tissue-specific increases in bioactive GAs that corresponded to the predominant native expression of the targeted paralogous gene pair. Although RNAi suppression of both paralogous pairs led to changes in wood development, they were much greater in the transgenics with suppressed PtGA2ox4 and PtGA2ox5. The degree of gene suppression in independent events was strongly associated with phenotypes, demonstrating dose-dependent control of growth by GA2ox RNA concentrations. • The expression and transgenic modifications reported here show that shoot- and leaf-expressed PtGA2ox4 and PtGA2ox5 specifically restrain aerial shoot growth, while root-expressed PtGA2ox2 and PtGA2ox7 promote root development. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust

Activation tagging is an effective gene tagging system in Populus

Knowledge of the functional relationship between genes and organismal phenotypes in perennial plants is extremely limited. Using a population of 627 independent events, we assessed the feasibility of activation tagging as a forward genetics tool for Populus. Mutant identification after 2 years of field testing was nearly sevenfold (6.5%) higher than in greenhouse studies that employed Arabidopsis and identical transformation vectors. Approximately two thirds of all mutant phenotypes were not seen in vitro and in the greenhouse; they were discovered only after the second year of field assessment. The trees\u27 large size (5-10 m in height), perennial growth, and interactions with the natural environment are factors that are thought to have contributed to the high rate of observable phenotypes in the field. The mutant phenotypes affected a variety of morphological and physiological traits, including leaf size and morphology, crown architecture, stature, vegetative dormancy, and tropic responses. Characterization of the insertion in more than 100 events with and without mutant phenotypes showed that tags predominantly (70%) inserted in a 13-Kbp region up- and downstream of the genes\u27 coding regions with approximately even distribution among the 19 chromosomes. Transcriptional activation was observed in many proximal genes studied. Successful phenotype recapitulation was observed in 10 of 12 retransformed genes tested, indicating true tagging and a functional relationship between the genes and observed phenotypes for most activation lines. Our studies indicate that in addition to associating mapping and QTL approaches, activation tagging can be used successfully as an effective forward gene discovery tool in Populus. © 2010 Springer-Verlag

Enhanced resistance to fungal pathogens in transgenic Populus tomentosa Carr. by overexpression of an nsLTP-like antimicrobial protein gene from motherwort (Leonurus japonicus)

The antimicrobial protein gene LJAMP2 is a plant non-specific lipid transfer protein from motherwort (Leonurus japonicus). In this study, it was introduced into Chinese white poplar (Populus tomentosa Carr.) via Agrobacterium-mediated transformation with neomycin phosphotransferase II gene conferring kanamycin resistance as selectable marker. A total of 16 poplar lines were obtained, and polymerase chain reaction (PCR) analysis established the stable integration of transgenes in the plant genome. Reverse transcription-PCR detected LJAMP2 expression in transgenic plants. Resistance to fungal pathogens Alternaria alternata (Fr.) Keissler and Colletotrichum gloeosporioides (Penz.) of transgenic poplar lines was tested. In vitro inhibitory activity against the fungal pathogens was evident from the crude leaf extracts from the transformants. In vivo assays showed that, after infection with both A. alternata (Fr.) Keissler and C. gloeosporioides (Penz.), there was a significant reduction in disease symptoms in transgenic poplar plants compared with the control. These results suggest that constitutive expression of the LJAMP2 gene from motherwort can be exploited to improve resistance to fungal pathogens in poplar. © The Author 2010. Published by Oxford University Press. All rights reserved

Gibberellins Regulate Lateral Root Formation in Populus through Interactions with Auxin and Other Hormones[C][W]

This work shows that gibberellins in poplar repress lateral root formation through interactions with other hormones like auxin. Gibberellin integrates aerial and root development, where aerial growth inhibition and concomitant stimulation of root proliferation produces a smaller plant with lower demands on environmental resources and a root system that can actively explore the soil environment

Field-grown miR156 transgenic switchgrass reproduction, yield, global gene expression analysis, and bioconfinement

Background Genetic engineering has been effective in altering cell walls for biofuel production in the bioenergy crop, switchgrass (Panicum virgatum). However, regulatory issues arising from gene flow may prevent commercialization of engineered switchgrass in the eastern United States where the species is native. Depending on its expression level, microRNA156 (miR156) can reduce, delay, or eliminate flowering, which may serve to decrease transgene flow. In this unique field study of transgenic switchgrass that was permitted to flower, two low (T14 and T35) and two medium (T27 and T37) miR156-overexpressing ‘Alamo’ lines with the transgene under the control of the constitutive maize (Zea mays) ubiquitin 1 promoter, along with nontransgenic control plants, were grown in eastern Tennessee over two seasons. Results miR156 expression was positively associated with decreased and delayed flowering in switchgrass. Line T27 did not flower during the 2-year study. Line T37 did flower, but not all plants produced panicles. Flowering was delayed in T37, resulting in 70.6% fewer flowers than controls during the second field year with commensurate decreased seed yield: 1205 seeds per plant vs. 18,539 produced by each control. These results are notable given that line T37 produced equivalent vegetative aboveground biomass to the controls. miR156 transcript abundance of field-grown plants was congruent with greenhouse results. The five miR156 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) target genes had suppressed expression in one or more of the transgenic lines. Line T27, which had the highest miR156 overexpression, showed significant downregulation for all five SPL genes. On the contrary, line T35 had the lowest miR156 overexpression and had no significant change in any of the five SPL genes. Conclusions Because of the research field’s geographical features, this study was the first instance of any genetically engineered trait in switchgrass, in which experimental plants were allowed to flower in the field in the eastern U.S.; USDA-APHIS-BRS regulators allowed open flowering. We found that medium overexpression of miR156, e.g., line T37, resulted in delayed and reduced flowering accompanied by high biomass production. We propose that induced miR156 expression could be further developed as a transgenic switchgrass bioconfinement tool to enable eventual commercialization