ABSTRACT
Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.
ABSTRACT
The production of biofuels and "green" chemicals from the lignocellulose of fast-growing hardwood species is hampered by extensive acetylation of xylan. Different strategies have been implemented to reduce xylan acetylation, resulting in transgenic plants that show good growth in the greenhouse, improved saccharification and fermentation, but the field performance of such plants has not yet been reported. The aim of this study was to evaluate the impact of reduced acetylation on field productivity and identify the best strategies for decreasing acetylation. Growth and biological stress data were evaluated for 18 hybrid aspen lines with 10-20% reductions in the cell wall acetyl content from a five year field experiment in Southern Sweden. The reduction in acetyl content was achieved either by suppressing the process of acetylation in the Golgi by reducing expression of REDUCED WALL ACETYLATION (RWA) genes, or by post-synthetic acetyl removal by fungal acetyl xylan esterases (AXEs) from two different families, CE1 and CE5, targeting them to cell walls. Transgene expression was regulated by either a constitutive promoter (35S) or a wood-specific promoter (WP). For the majority of transgenic lines, growth was either similar to that in WT and transgenic control (WP:GUS) plants, or slightly reduced. The slight reduction was observed in the AXE-expressing lines regulated by the 35S promoter, not those with the WP promoter which limits expression to cells developing secondary walls. Expressing AXEs regulated by the 35S promoter resulted in increased foliar arthropod chewing, and altered condensed tannins and salicinoid phenolic glucosides (SPGs) profiles. Greater growth inhibition was observed in the case of CE5 than with CE1 AXE, and it was associated with increased foliar necrosis and distinct SPG profiles, suggesting that CE5 AXE could be recognized by the pathogen-associated molecular pattern system. For each of three different constructs, there was a line with dwarfism and growth abnormalities, suggesting random genetic/epigenetic changes. This high frequency of dwarfism (17%) is suggestive of a link between acetyl metabolism and chromatin function. These data represent the first evaluation of acetyl-reduced plants from the field, indicating some possible pitfalls, and identifying the best strategies, when developing highly productive acetyl-reduced feedstocks.