Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

Field evaluation of transgenic hybrid poplars with desirable wood properties and enhanced growth for biofuel production by bicistronic expression of PdGA20ox1 and PtrMYB3 in wood-forming tissue

Background To create an ideotype woody bioenergy crop with desirable growth and biomass properties, we utilized the viral 2A-meidated bicistronic expression strategy to express both PtrMYB3 (MYB46 ortholog of Populus trichocarpa, a master regulator of secondary wall biosynthesis) and PdGA20ox1 (a GA20-oxidase from Pinus densiflora that produces gibberellins) in wood-forming tissue (i.e., developing xylem). Results Transgenic Arabidopsis plants expressing the gene construct DX15::PdGA20ox1-2A-PtrMYB3 showed a significant increase in both stem fresh weight (threefold) and secondary wall thickening (1.27-fold) relative to wild-type (WT) plants. Transgenic poplars harboring the same gene construct grown in a greenhouse for 60 days had a stem fresh weight up to 2.6-fold greater than that of WT plants. In a living modified organism (LMO) field test conducted for 3 months of active growing season, the stem height and diameter growth of the transgenic poplars were 1.7- and 1.6-fold higher than those of WT plants, respectively, with minimal adverse growth defects. Although no significant changes in secondary wall thickening of the stem tissue of the transgenic poplars were observed, cellulose content was increased up to 14.4 wt% compared to WT, resulting in improved saccharification efficiency of the transgenic poplars. Moreover, enhanced woody biomass production by the transgenic poplars was further validated by re-planting in the same LMO field for additional two growing seasons. Conclusions Taken together, these results show considerably enhanced wood formation of our transgenic poplars, with improved wood quality for biofuel production.

Effects of Bt-Cry1Ah1 Transgenic Poplar on Target and Non-Target Pests and Their Parasitic Natural Enemy in Field and Laboratory Trials

Increasing areas of artificial afforestation and poplar monoculture in China have led to serious problems with insect pests. The development of genetic engineering technology, such as transgenic modification with Bacillus thuringiensis (Bt) genes, provides novel solutions to the pest problem. We generated a Bt-Cry1Ah1 gene incorporating codon optimization and transferred it into Populus deltoides × P. euramericana cv “Nanlin895” using an Agrobacterium-mediated method. The resulting Bt-Cry1Ah1 transgenic poplars were planted in the field with permission from the State Forestry Administration in 2017. Field and laboratory studies were conducted in Jiangsu, China, to investigate the effects of these transgenic poplars expressing the Cry1Ah1 protein on target and non-target pests and their parasitic natural enemy. Target pests included Hyphantria cunea (Lepidoptera, Arctiidae), Micromelalopha troglodyta (Lepidoptera, Notodontidae), and Clostera anachoreta (Lepidoptera, Notodontidae). Plagiodera versicolora (Coleoptera, Chrysomelidae) served as the non-target pest. Laboratory trials showed that the six transgenic poplar lines exhibited resistance against the target insects. The corrected mortality rates of the target pest larvae fed leaves from the six lines were as high as 87.0%, significantly higher than that of the control. However, the corrected mortality rate of the non-target pest larvae was markedly lower and did not differ significantly from that of the control. Field experiments showed that transgenic poplar exhibited resistance against H. cunea and M. troglodyta. Field mortality rates were slightly higher than laboratory mortality rates. In addition, we investigated Chouioia cunea (Hymenoptera, Eulophidae) as a parasitoid of H. cunea pupae that had been fed transgenic poplar leaves. The emergence time, parasitism rate, and abundance of C. cunea did not differ significantly from those of the control. Therefore, Bt-Cry1Ah1 transgenic poplar can be used to effectively control damage by target insect pests without negatively affecting non-target insects and parasitoids.

Cross-talk between the methylerythritol phosphate and mevalonic acid pathways of isoprenoid biosynthesis in poplar

Plants use two distinct isoprenoid biosynthesis pathways: the methylerythritol phosphate (MEP) pathway and mevalonic acid (MVA) pathway. 1-deoxy-D-xylulose5-phosphate synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) are the rate-limiting enzymes in the MEP pathway, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key regulatory enzyme in the MVA pathway. Previously, overexpression of Populus trichocarpa PtDXR in Nanlin 895 poplar was found to upregulate resistance to salt and drought stresses, and the transgenic poplars showed improved growth. In the present study, PtHMGR overexpressors (OEs) exhibited higher expression levels of DXS, DXR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) and lower expression levels of 2-C-methyl-d-erythritol4-phosphate cytidylyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), and 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) than non-transgenic poplars (NT). However, the poplar PtDXR-OEs showed upregulated expression levels of MEP-related genes and downregulated expression of MVA-related genes. Moreover, overexpression of PtDXR and PtHMGR in poplars caused changes in MVA-derived trans-zeatin-riboside (TZR), isopentenyl adenosine (IPA), castasterone (CS) and 6-deoxocastasterone (DCS), as well as MEP-derived carotenoids, gibberellins (GAs), and abscisic acid (ABA). In PtHMGR-OEs, greater accumulation of geranyl diphosphate synthase (GPS) and geranyl pyrophosphate synthase (GPPS) transcript levels in the MEP pathway led to accumulation of MEP-derived isoprenoids, while upregulation of farnesyl diphosphate synthase (FPS) expression in the MVA pathway contributed to increased levels of MVA-derived isoprenoids. Similarly, in PtDXR-OEs, increased GPS and GPPS transcript levels in the MEP pathway boosted MEP-derived isoprenoid levels and changes in FPS expression affected MVA-derived isoprenoid yields. From these results, we can conclude that cross-talk exists between the MVA and MEP pathways.

Characterization, expression profiling, and functional analysis of a Populus trichocarpa defensin gene and its potential as an anti-Agrobacterium rooting medium additive

The diverse antimicrobial properties of defensins have attracted wide scientific interest in recent years. Also, antimicrobial peptides (AMPs), including cecropins, histatins, defensins, and cathelicidins, have recently become an antimicrobial research hotspot for their broad-spectrum antibacterial and antifungal activities. In addition, defensins play important roles in plant growth, development, and physiological metabolism, and demonstrate tissue specificity and regulation in response to pathogen attack or abiotic stress. In this study, we performed molecular cloning, characterization, expression profiling, and functional analysis of a defensin from Populus trichocarpa. The PtDef protein was highly expressed in the prokaryotic Escherichia coli system as a fusion protein (TrxA–PtDef). The purified protein exhibited strong antibacterial and antifungal functions. We then applied PtDef to rooting culture medium as an alternative exogenous additive to cefotaxime. PtDef expression levels increased significantly following both biotic and abiotic treatment. The degree of leaf damage observed in wild-type (WT) and transgenic poplars indicates that transgenic poplars that overexpress the PtDef gene gain enhanced disease resistance to Septotis populiperda. To further study the salicylic acid (SA) and jasmonic acid (JA) signal transduction pathways, SA- and JA-related and pathogenesis-related genes were analyzed using quantitative reverse-transcription polymerase chain reaction; there were significant differences in these pathways between transgenic and WT poplars. The defensin from Populus trichocarpa showed significant activity of anti-bacteria and anti-fungi. According to the results of qRT-PCR and physiological relevant indicators, the applied PtDef to rooting culture medium was chosen as an alternative exogenous additive to cefotaxime. Overexpressing the PtDef gene in poplar improve the disease resistance to Septotis populiperda.