Downregulation of barley ferulate 5-hydroxylase dramatically alters straw lignin structure without impact on mechanical properties

Abstract

Barley is a major cereal crop for temperate climates, and a diploid genetic model for polyploid wheat. Cereal straw biomass is an attractive source of feedstock for green technologies but lignin, a key determinant of feedstock recalcitrance, complicates bio-conversion processes. However, manipulating lignin content to improve the conversion process could negatively affect agronomic traits. An alternative approach is to manipulate lignin composition which influences the physical and chemical properties of straw. This study validates the function of a barley ferulate 5-hydroxylase gene and demonstrates that its downregulation using the RNA-interference approach substantially impacts lignin composition. We identified five barley genes having putative ferulate 5-hydroxylase activity. Downregulation of HvF5H1 substantially reduced the lignin syringyl/guaiacyl (S/G) ratio in straw while the lignin content, straw mechanical properties, plant growth habit, and grain characteristics all remained unaffected. Metabolic profiling revealed significant changes in the abundance of 173 features in the HvF5H1-RNAi lines. The drastic changes in the lignin polymer of transgenic lines highlight the plasticity of barley lignification processes and the associated potential for manipulating and improving lignocellulosic biomass as a feedstock for green technologies. On the other hand, our results highlight some differences between the lignin biosynthetic pathway in barley, a temperate climate grass, and the warm climate grass, rice, and underscore potential diversity in the lignin biosynthetic pathways in grasses.

ShF5H1 overexpression increases syringyl lignin and improves saccharification in sugarcane leaves

“…The blastp search in the sugarcane genome database S. officinarum x spontaneum R570 v2.1 (Phytozome 13) allowed us to retrieve the top sixteen protein sequences (e-values: 0 - 18e-180), compared to the previously identified 50 , 52 and cloned (KT777465.1) ShF5H. The amino acid (aa) sequences of sugarcane, monocot species, and Arabidopsis, retrieved from the Phytozome and TAIR databases using ShF5H1 as a query (BLASTp), along with the sequences from Shafiei et al, 44 were aligned and used to construct the phylogenetic tree (Figure S3). The CYP84 family comprises cytochrome P450 (CYP) monooxygenase enzymes, as F5H from the lignin pathway.…”

“…The major group is divided into two subgroups: one containing non-characterized proteins, which includes TaF5H2–3, HvF5H4, MsF5H3, SbF5H2, and ShF5H2–4. In contrast, the red-colored subgroup comprises grass proteins that have been functionally characterized (indicated by single asterisks), such as SbF5H1, 43 OsF5H1, 42 HvF5H1, 44 and PvF5H1a, 31 suggesting that the members of this subgroup play a similar role in S-lignin synthesis. Among the sugarcane CYP84A proteins, only one member belongs to the mentioned subgroup, ShF5H1 (indicated by double asterisks), positioned closest to SbF5H, whose overexpression led to an increase in syringyl acid and S units in sorghum.…”

“…Additionally, previously reported sequences were included in the phylogenetic analysis: PvF5H1a (GenBank accession number BAO37287.1) 31 and barley (Hv), wheat (Ta), Brachypodium (Bd), Miscanthus (Ms), green foxtail (Sv), Selaginella moellenodorffii (Sm). 44 The amino acid sequences (aa) were aligned in the ClustalX v2 program. 53 The evolutionary history was inferred using the Maximum Likelihood method and Whelan and Goldman model.…”

“…Poplar 30 and Arabidopsis 40 , 41 showed enhanced digestibility when the S/G ratio was increased by F5H overexpression. In monocots, F5H was identified and characterized in rice, 42 switchgrass, 31 sorghum 43 and barley, 44 and its counterpart function on the S branch was confirmed but still lacked a demonstration of digestibility improvement. It has been reported that CAld5H (coniferaldehyde 5 hydroxylase) has a different activity compared to F5H as the former is more specific to the downstream pathway.…”

Lignin Biosynthesis and Its Diversified Roles in Disease Resistance

“…However, a serial of work from the transgenic plants, feeding tests, and in vitro biochemistry have demonstrated that F5H actually used coniferaldehyde and coniferyl alcohol to form 5-hydro-coniferaldehyde and 5-hydro-coniferyl alcohol then syringyl alcohol. Over-expressing F5H in transgenic tobacco and poplar gave rise to lignin that was composed of almost S units [ 26 , 27 , 28 ]. Therefore, F5H is now also called coniferaldehyde 5-hydroxylase (CAld-5H) [ 29 ].…”