Suppression of the Arabidopsis cinnamoyl-CoA reductase 1-6 intronic T-DNA mutation by epigenetic modification.

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA-containing intron. Consequently, the suppressed ccr1-6 allele was named epiccr1-6. Long-read sequencing revealed that epiccr1-6, not ccr1-6, carries dense cytosine methylation over the full length of the T-DNA. We showed that the SAIL T-DNA in the UGT72E3 locus could trigger the trans T-DNA suppression of the GABI-Kat T-DNA in the CCR1 locus. Furthermore, we scanned the literature for other potential cases of trans T-DNA suppression in Arabidopsis and found that 22% of the publications matching our query report on double or higher-order T-DNA mutants that meet the minimal requirements for trans T-DNA suppression. These combined observations indicate that intronic T-DNA mutants need to be used with caution since methylation of intronic T-DNA might derepress gene expression and can thereby confound results.

Whole genome duplication of wild-type and CINNAMYL ALCOHOL DEHYDROGENASE1-downregulated hybrid poplar reduces biomass yield and causes a brittle apex phenotype in field-grown wild types.

The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabidopsis tetraploids, an increase in biomass yield was accompanied by a reduction in lignin content and, as a result, a higher saccharification efficiency was achieved compared with diploid controls. Here, we evaluated whether the results obtained in Arabidopsis could be translated into poplar and whether the enhanced saccharification yield upon alkaline pretreatment of hairpin-downregulated CINNAMYL ALCOHOL DEHYDROGENASE1 (hpCAD) transgenic poplar could be further improved upon a whole genome duplication. Using a colchicine treatment, wild-type (WT) Populus tremula x P. alba cv. INRA 717-1B4, a commonly used model clone in tree biotechnology research, and hpCAD tetraploids were generated and grown in the greenhouse. In parallel, WT tetraploid poplars were grown in the field. In contrast to Arabidopsis, a whole genome duplication of poplar had a negative impact on the biomass yield of both greenhouse- and field-grown trees. Strikingly, field-grown WT tetraploids developed a brittle apex phenotype, i.e., their tip broke off just below the apex. In addition, the chromosome doubling altered the biomass composition of field-grown, but not of greenhouse-grown tetraploid poplars. More specifically, the lignin content of field-grown tetraploid poplars was increased at the expense of matrix polysaccharides. This increase in lignin deposition in biomass is likely the cause of the observed brittle apex phenotype, though no major differences in stem anatomy or in mechanical properties could be found between di- and tetraploid WT poplars grown in the field. Finally, without biomass pretreatment, the saccharification efficiency of greenhouse- and field-grown WT diploids was not different from that of tetraploids, whereas that of greenhouse-grown hpCAD tetraploids was higher than that of greenhouse-grown diploids. Upon alkaline pretreatment, the saccharification yield of diploids was similar to that of tetraploids for all genotypes and growth conditions tested. This study showed that a whole genome duplication in hybrid WT and hpCAD poplar did neither result in further improvements in biomass yield, nor in improved biomass composition and, hence, saccharification performance.

Field and saccharification performances of poplars severely downregulated in CAD1.

Lignin is one of the main factors causing lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Glasshouse-grown poplars severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE 1 (CAD1), the enzyme catalysing the last step in the monolignol-specific branch of lignin biosynthesis, have increased saccharification yields and normal growth. Here, we assess the performance of these hpCAD poplars in the field under short rotation coppice culture for two consecutive rotations of 1 yr and 3 yr. While 1-yr-old hpCAD wood had 10% less lignin, 3-yr-old hpCAD wood had wild-type lignin levels. Because of their altered cell wall composition, including elevated levels of cinnamaldehydes, both 1-yr-old and 3-yr-old hpCAD wood showed enhanced saccharification yields upon harsh alkaline pretreatments (up to +85% and +77%, respectively). In contrast with previous field trials with poplars less severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD), the hpCAD poplars displayed leaning phenotypes, early bud set, early flowering and yield penalties. Moreover, hpCAD wood had enlarged vessels, decreased wood density and reduced relative and free water contents. Our data show that the phenotypes of CAD-deficient poplars are strongly dependent on the environment and underpin the importance of field trials in translating basic research towards applications.

Overexpression of the scopoletin biosynthetic pathway enhances lignocellulosic biomass processing.

Lignin is the main factor limiting the enzymatic conversion of lignocellulosic biomass into fermentable sugars. To reduce the recalcitrance engendered by the lignin polymer, the coumarin scopoletin was incorporated into the lignin polymer through the simultaneous expression of FERULOYL-CoA 6'-HYDROXYLASE 1 (F6'H1) and COUMARIN SYNTHASE (COSY) in lignifying cells in Arabidopsis. The transgenic lines overproduced scopoletin and incorporated it into the lignin polymer, without adversely affecting plant growth. About 3.3% of the lignin units in the transgenic lines were derived from scopoletin, thereby exceeding the levels of the traditional p-hydroxyphenyl units. Saccharification efficiency of alkali-pretreated scopoletin-overproducing lines was 40% higher than for wild type.

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar.

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as 'good neighbors' in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of 'good neighbors' in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.