A large-scale forward genetic screen for maize mutants with altered lignocellulosic properties.

The development of efficient pipelines for the bioconversion of grass lignocellulosic feedstocks is challenging due to the limited understanding of the molecular mechanisms controlling the synthesis, deposition, and degradation of the varying polymers unique to grass cell walls. Here, we describe a large-scale forward genetic approach resulting in the identification of a collection of chemically mutagenized maize mutants with diverse alterations in their cell wall attributes such as crystalline cellulose content or hemicellulose composition. Saccharification yield, i.e. the amount of lignocellulosic glucose (Glc) released by means of enzymatic hydrolysis, is increased in two of the mutants and decreased in the remaining six. These mutants, termed candy-leaf (cal), show no obvious plant growth or developmental defects despite associated differences in their lignocellulosic composition. The identified cal mutants are a valuable tool not only to understand recalcitrance of grass lignocellulosics to enzymatic deconstruction but also to decipher grass-specific aspects of cell wall biology once the genetic basis, i.e. the location of the mutation, has been identified.

Glycosylphosphatidylinositol anchor lipid remodeling directs proteins to the plasma membrane and governs cell wall mechanics.

Glycosylphosphatidylinositol (GPI) anchoring is a common protein modification that targets proteins to the plasma membrane (PM). Knowledge about the GPI lipid tail, which guides the secretion of GPI-anchored proteins (GPI-APs), is limited in plants. Here, we report that rice (Oryza sativa) BRITTLE CULM16 (BC16), a membrane-bound O-acyltransferase (MBOAT) remodels GPI lipid tails and governs cell wall biomechanics. The bc16 mutant exhibits fragile internodes, resulting from reduced cell wall thickness and cellulose content. BC16 is the only MBOAT in rice and is located in the endoplasmic reticulum and Golgi apparatus. Yeast gup1Δ mutant restoring assay and GPI lipid composition analysis demonstrated BC16 as a GPI lipid remodelase. Loss of BC16 alters GPI lipid structure and disturbs the targeting of BC1, a GPI-AP for cellulose biosynthesis, to the PM lipid nanodomains. Atomic force microscopy revealed compromised deposition of cellulosic nanofibers in bc16, leading to an increased Young's modulus and abnormal mechanical properties. Therefore, BC16-mediated lipid remodeling directs the GPI-APs, such as BC1, to the cell surface to fulfill multiple functions, including cellulose organization. Our work unravels a mechanism by which GPI lipids are remodeled in plants and provides insights into the control of cell wall biomechanics, offering a tool for breeding elite crops with improved support strength.

MYB61 is regulated by GRF4 and promotes nitrogen utilization and biomass production in rice.

Nitrogen (N) is a macronutrient that boosts carbon (C) metabolism and plant growth leading to biomass accumulation. The molecular connection between nitrogen utilization efficiency (NUE) and biomass production remains unclear. Here, via quantitative trait loci analysis and map-based cloning, we reveal that natural variation at the MYB61 locus leads to differences in N use and cellulose biogenesis between indica and japonica subspecies of rice. MYB61, a transcriptional factor that regulates cellulose synthesis, is directly regulated by a known NUE regulator GROWTH-REGULATING FACTOR4 (GRF4), which coordinates cellulosic biomass production and N utilization. The variation at MYB61 has been selected during indica and japonica domestication. The indica allele of MYB61 displays robust transcription resulting in higher NUE and increased grain yield at reduced N supply than that of japonica. Our study hence unravels how C metabolism is linked to N uptake and may provide an opportunity to reduce N use for sustainable agriculture.

Rice Homeobox Protein KNAT7 Integrates the Pathways Regulating Cell Expansion and Wall Stiffness.

During growth, plant cells must coordinate cell expansion and cell wall reinforcement by integrating distinct regulatory pathways in concert with intrinsic and external cues. However, the mechanism underpinning this integration is unclear, as few of the regulators that orchestrate cell expansion and wall strengthening have been identified. Here, we report a rice (Oryza sativa) Class II KNOX-like homeobox protein, KNOTTED ARABIDOPSIS THALIANA7 (KNAT7), that interacts with different partners to govern cell expansion and wall thickening. A loss-of-function mutation in KNAT7 enhanced wall mechanical strength and cell expansion, resulting in improved lodging resistance and grain size. Overexpression of KNAT7 gave rise to the opposite phenotypes, with plants having weaker cell walls and smaller grains. Biochemical and gene expression analyses revealed that rice KNAT7 interacts with a secondary wall key regulator, NAC31, and a cell growth master regulator, Growth-Regulating Factor 4 (GRF4). The KNAT7-NAC31 and KNAT7-GRF4 modules suppressed regulatory pathways of cell expansion and wall reinforcement, as we show in internode and panicle development. These modules function in sclerenchyma fiber cells and modulate fiber cell length and wall thickness. Hence, our study uncovers a mechanism for the combined control of cell size and wall strengthening, providing a tool to improve lodging resistance and yield in rice production.

Arabinosyl Deacetylase Modulates the Arabinoxylan Acetylation Profile and Secondary Wall Formation.

Acetylation, a prevalent modification of cell-wall polymers, is a tightly controlled regulatory process that orchestrates plant growth and environmental adaptation. However, due to limited characterization of the enzymes involved, it is unclear how plants establish and dynamically regulate the acetylation pattern in response to growth requirements. In this study, we identified a rice (Oryza sativa) GDSL esterase that deacetylates the side chain of the major rice hemicellulose, arabinoxylan. Acetyl esterases involved in arabinoxylan modification were screened using enzymatic assays combined with mass spectrometry analysis. One candidate, DEACETYLASE ON ARABINOSYL SIDECHAIN OF XYLAN1 (DARX1), is specific for arabinosyl residues. Disruption of DARX1 via Tos17 insertion and CRISPR/Cas9 approaches resulted in the accumulation of acetates on the xylan arabinosyl side chains. Recombinant DARX1 abolished the excess acetyl groups on arabinoxylan-derived oligosaccharides of the darx1 mutants in vitro. Moreover, DARX1 is localized to the Golgi apparatus. Two-dimensional 13C-13C correlation spectroscopy and atomic force microscopy further revealed that the abnormal acetylation pattern observed in darx1 interrupts arabinoxylan conformation and cellulose microfibril orientation, resulting in compromised secondary wall patterning and reduced mechanical strength. This study provides insight into the mechanism controlling the acetylation pattern on arabinoxylan side chains and suggests a strategy to breed robust elite crops.

A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice.

Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth.