Grass secondary cell walls, Brachypodium distachyon as a model for discovery.

A key aspect of plant growth is the synthesis and deposition of cell walls. In specific tissues and cell types including xylem and fibre, a thick secondary wall comprised of cellulose, hemicellulose and lignin is deposited. Secondary cell walls provide a physical barrier that protects plants from pathogens, promotes tolerance to abiotic stresses and fortifies cells to withstand the forces associated with water transport and the physical weight of plant structures. Grasses have numerous cell wall features that are distinct from eudicots and other plants. Study of the model species Brachypodium distachyon as well as other grasses has revealed numerous features of the grass cell wall. These include the characterisation of xylosyl and arabinosyltransferases, a mixed-linkage glucan synthase and hydroxycinnamate acyltransferases. Perhaps the most fertile area for discovery has been the formation of lignins, including the identification of novel substrates and enzyme activities towards the synthesis of monolignols. Other enzymes function as polymerising agents or transferases that modify lignins and facilitate interactions with polysaccharides. The regulatory aspects of cell wall biosynthesis are largely overlapping with those of eudicots, but salient differences among species have been resolved that begin to identify the determinants that define grass cell walls.

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation.

Plants are continuously exposed to diurnal fluctuations in light and temperature, and spontaneous changes in their physical or biotic environment. The circadian clock coordinates regulation of gene expression with a 24 h period, enabling the anticipation of these events. We used RNA sequencing to characterize the Brachypodium distachyon transcriptome under light and temperature cycles, as well as under constant conditions. Approximately 3% of the transcriptome was regulated by the circadian clock, a smaller proportion than reported in most other species. For most transcripts that were rhythmic under all conditions, including many known clock genes, the period of gene expression lengthened from 24 to 27 h in the absence of external cues. To functionally characterize the cyclic transcriptome in B. distachyon, we used Gene Ontology enrichment analysis, and found several terms significantly associated with peak expression at particular times of the day. Furthermore, we identified sequence motifs enriched in the promoters of similarly phased genes, some potentially associated with transcription factors. When considering the overlap in rhythmic gene expression and specific pathway behavior, thermocycles was the prevailing cue that controlled diurnal gene regulation. Taken together, our characterization of the rhythmic B. distachyon transcriptome represents a foundational resource with implications in other grass species.

SECONDARY WALL ASSOCIATED MYB1 is a positive regulator of secondary cell wall thickening in Brachypodium distachyon and is not found in the Brassicaceae.

Grass biomass is comprised chiefly of secondary walls that surround fiber and xylem cells. A regulatory network of interacting transcription factors in part regulates cell wall thickening. We identified Brachypodium distachyon SECONDARY WALL ASSOCIATED MYB1 (SWAM1) as a potential regulator of secondary cell wall biosynthesis based on gene expression, phylogeny, and transgenic plant phenotypes. SWAM1 interacts with cellulose and lignin gene promoters with preferential binding to AC-rich sequence motifs commonly found in the promoters of cell wall-related genes. SWAM1 overexpression (SWAM-OE) lines had greater above-ground biomass with only a slight change in flowering time while SWAM1 dominant repressor (SWAM1-DR) plants were severely dwarfed with a striking reduction in lignin of sclerenchyma fibers and stem epidermal cell length. Cellulose, hemicellulose, and lignin genes were significantly down-regulated in SWAM1-DR plants and up-regulated in SWAM1-OE plants. There was no reduction in bioconversion yield in SWAM1-OE lines; however, it was significantly increased for SWAM1-DR samples. Phylogenetic and syntenic analyses strongly suggest that the SWAM1 clade was present in the last common ancestor between eudicots and grasses, but is not in the Brassicaceae. Collectively, these data suggest that SWAM1 is a transcriptional activator of secondary cell wall thickening and biomass accumulation in B. distachyon.

A stress-associated protein, AtSAP13, from Arabidopsis thaliana provides tolerance to multiple abiotic stresses.

Members of Stress-Associated Protein (SAP) family in plants have been shown to impart tolerance to multiple abiotic stresses, however, their mode of action in providing tolerance to multiple abiotic stresses is largely unknown. There are 14 SAP genes in Arabidopsis thaliana containing A20, AN1, and Cys2-His2 zinc finger domains. AtSAP13, a member of the SAP family, carries two AN1 zinc finger domains and an additional Cys2-His2 domain. AtSAP13 transcripts showed upregulation in response to Cd, ABA, and salt stresses. AtSAP13 overexpression lines showed strong tolerance to toxic metals (AsIII, Cd, and Zn), drought, and salt stress. Further, transgenic lines accumulated significantly higher amounts of Zn, but less As and Cd accumulation in shoots and roots. AtSAP13 promoter-GUS fusion studies showed GUS expression predominantly in the vascular tissue, hydathodes, and the apical meristem and region of root maturation and elongation as well as the root hairs. At the subcellular level, the AtSAP13-eGFP fusion protein was found to localize in both nucleus and cytoplasm. Through yeast one-hybrid assay, we identified several AP2/EREBP family transcription factors that interacted with the AtSAP13 promoter. AtSAP13 and its homologues will be highly useful for developing climate resilient crops.

Climate-smart crops with enhanced photosynthesis.

The potential of enhanced photosynthetic efficiency to help achieve the sustainable yield increases required to meet future demands for food and energy has spurred intense research towards understanding, modeling, and engineering photosynthesis. These current efforts, largely focused on the C3 model Arabidopsis thaliana or crop plants (e.g. rice, sorghum, maize, and wheat), could be intensified and broadened using model systems closely related to our food, feed, and energy crops and that allow rapid design-build-test-learn cycles. In this outlooking Opinion, we advocate for a concerted effort to expand our understanding and improve our ability to redesign carbon uptake, allocation, and utilization. We propose two specific research directions that combine enhanced photosynthesis with climate-smart metabolic attributes: (i) engineering pathways for flexible (facultative) C3-C4 metabolism where plants will operate either C3 or C4 photosynthesis based on environmental conditions such as temperature, light, and atmospheric CO2 levels; and (ii) increasing rhizospheric sink strength for carbon utilization, including strategies that allow for augmented transport of carbon to the soil for improved soil properties and carbon storage without jeopardizing aboveground crop biomass. We argue that such ambitious undertakings be first approached and demonstrated by exploring the full genomic potential of two model grasses, the C3Brachypodium distachyon and the C4Setaria viridis. The development of climate-smart crops could provide novel and bold solutions to increase crop productivity while reducing atmospheric carbon and nitrogen emissions.

A cell wall reference profile for Miscanthus bioenergy crops highlights compositional and structural variations associated with development and organ origin.

Miscanthus spp. are promising lignocellulosic energy crops, but cell wall recalcitrance to deconstruction still hinders their widespread use as bioenergy and biomaterial feedstocks. Identification of cell wall characteristics desirable for biorefining applications is crucial for lignocellulosic biomass improvement. However, the task of scoring biomass quality is often complicated by the lack of a reference for a given feedstock. A multidimensional cell wall analysis was performed to generate a reference profile for leaf and stem biomass from several miscanthus genotypes harvested at three developmentally distinct time points. A comprehensive suite of 155 monoclonal antibodies was used to monitor changes in distribution, structure and extractability of noncellulosic cell wall matrix glycans. Glycan microarrays complemented with immunohistochemistry elucidated the nature of compositional variation, and in situ distribution of carbohydrate epitopes. Key observations demonstrated that there are crucial differences in miscanthus cell wall glycomes, which may impact biomass amenability to deconstruction. For the first time, variations in miscanthus cell wall glycan components were comprehensively characterized across different harvests, organs and genotypes, to generate a representative reference profile for miscanthus cell wall biomass. Ultimately, this portrait of the miscanthus cell wall will help to steer breeding and genetic engineering strategies for the development of superior energy crops.

Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates.

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range.

UNLABELLED: • PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses (B. distachyon, B. stacei) and their derived allotetraploid (B. hybridum), native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identified potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range.• METHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene flow among the hybridizing species of the complex.• KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth.• CONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversification was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

Genotype, development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus.

BACKGROUND AND AIMS: Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock. METHODS: Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol. KEY RESULTS: Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent. CONCLUSIONS: It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene-trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.

Shoring up the base: the development and regulation of cortical sclerenchyma in grass nodal roots.

Plants depend on the combined action of a shoot-root-soil system to maintain their anchorage to the soil. Mechanical failure of any component of this system results in lodging, a permanent and irreversible inability to maintain vertical orientation. Models of anchorage in grass crops identify the compressive strength of roots near the soil surface as key determinant of resistance to lodging. Indeed, studies of disparate grasses report a ring of thickened, sclerenchyma cells surrounding the root cortex, present only at the base of nodal roots. Here, in the investigation of the development and regulation of this agronomically important trait, we show that development of these cells is uncoupled from the maturation of other secondary cell wall-fortified cells, and that cortical sclerenchyma wall thickening is stimulated by mechanical forces transduced from the shoot to the root. We also show that exogenous application of gibberellic acid stimulates thickening of lignified cell types in the root, including cortical sclerenchyma, but is not sufficient to establish sclerenchyma identity in cortex cells. Leveraging the ability to manipulate cortex development via mechanical stimulus, we show that cortical sclerenchyma development alters root mechanical properties and improves resistance to lodging. We describe transcriptome changes associated with cortical sclerenchyma development under both ambient and mechanically stimulated conditions and identify SECONDARY WALL NAC7 as a putative regulator of mechanically responsive cortex cell wall development at the root base.

Mechanically induced localisation of SECONDARY WALL INTERACTING bZIP is associated with thigmomorphogenic and secondary cell wall gene expression.

Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

BACKGROUND: Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. RESULTS: We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. CONCLUSION: These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.

Perturbation of Brachypodium distachyon CELLULOSE SYNTHASE A4 or 7 results in abnormal cell walls.

BACKGROUND: Cellulose is an integral component of the plant cell wall and accounts for approximately forty percent of total plant biomass but understanding its mechanism of synthesis remains elusive. CELLULOSE SYNTHASE A (CESA) proteins function as catalytic subunits of a rosette-shaped complex that synthesizes cellulose at the plasma membrane. Arabidopsis thaliana and rice (Oryza sativa) secondary wall CESA loss-of-function mutants have weak stems and irregular or thin cell walls. RESULTS: Here, we identify candidates for secondary wall CESAs in Brachypodium distachyon as having similar amino acid sequence and expression to those characterized in A. thaliana, namely CESA4/7/8. To functionally characterize BdCESA4 and BdCESA7, we generated loss-of-function mutants using artificial microRNA constructs, specifically targeting each gene driven by a maize (Zea mays) ubiquitin promoter. Presence of the transgenes reduced BdCESA4 and BdCESA7 transcript abundance, as well as stem area, cell wall thickness of xylem and fibers, and the amount of crystalline cellulose in the cell wall. CONCLUSION: These results suggest BdCESA4 and BdCESA7 play a key role in B. distachyon secondary cell wall biosynthesis.

Brachypodium: 20 years as a grass biology model system; the way forward?

It has been 20 years since Brachypodium distachyon was suggested as a model grass species, but ongoing research now encompasses the entire genus. Extensive Brachypodium genome sequencing programmes have provided resources to explore the determinants and drivers of population diversity. This has been accompanied by cytomolecular studies to make Brachypodium a platform to investigate speciation, polyploidisation, perenniality, and various aspects of chromosome and interphase nucleus organisation. The value of Brachypodium as a functional genomic platform has been underscored by the identification of key genes for development, biotic and abiotic stress, and cell wall structure and function. While Brachypodium is relevant to the biofuel industry, its impact goes far beyond that as an intriguing model to study climate change and combinatorial stress.

A morning-specific phytohormone gene expression program underlying rhythmic plant growth.

Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.

Network discovery pipeline elucidates conserved time-of-day-specific cis-regulatory modules.

Correct daily phasing of transcription confers an adaptive advantage to almost all organisms, including higher plants. In this study, we describe a hypothesis-driven network discovery pipeline that identifies biologically relevant patterns in genome-scale data. To demonstrate its utility, we analyzed a comprehensive matrix of time courses interrogating the nuclear transcriptome of Arabidopsis thaliana plants grown under different thermocycles, photocycles, and circadian conditions. We show that 89% of Arabidopsis transcripts cycle in at least one condition and that most genes have peak expression at a particular time of day, which shifts depending on the environment. Thermocycles alone can drive at least half of all transcripts critical for synchronizing internal processes such as cell cycle and protein synthesis. We identified at least three distinct transcription modules controlling phase-specific expression, including a new midnight specific module, PBX/TBX/SBX. We validated the network discovery pipeline, as well as the midnight specific module, by demonstrating that the PBX element was sufficient to drive diurnal and circadian condition-dependent expression. Moreover, we show that the three transcription modules are conserved across Arabidopsis, poplar, and rice. These results confirm the complex interplay between thermocycles, photocycles, and the circadian clock on the daily transcription program, and provide a comprehensive view of the conserved genomic targets for a transcriptional network key to successful adaptation.

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors.

Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution.

A high-resolution map of Arabidopsis recombinant inbred lines by whole-genome exon array hybridization.

Recombinant populations were the basis for Mendel's first genetic experiments and continue to be key to the study of genes, heredity, and genetic variation today. Genotyping several hundred thousand loci in a single assay by hybridizing genomic DNA to oligonucleotide arrays provides a powerful technique to improve precision linkage mapping. The genotypes of two accessions of Arabidopsis were compared by using a 400,000 feature exon-specific oligonucleotide array. Around 16,000 single feature polymorphisms (SFPs) were detected in approximately 8,000 of the approximately 26,000 genes represented on the array. Allelic variation at these loci was measured in a recombinant inbred line population, which defined the location of 815 recombination breakpoints. The genetic linkage map had a total length of 422.5 cM, with 676 informative SFP markers representing intervals of approximately 0.6 cM. One hundred fifteen single gene intervals were identified. Recombination rate, SFP distribution, and segregation in this population are not uniform. Many genomic regions show a clustering of recombination events including significant hot spots. The precise haplotype structure of the recombinant population was defined with unprecedented accuracy and resolution. The resulting linkage map allows further refinement of the hundreds of quantitative trait loci identified in this well-studied population. Highly variable recombination rates along each chromosome and extensive segregation distortion were observed in the population.

Regulation of Cell Wall Thickening by a Medley of Mechanisms.

To provide physical support for developing structures and to withstand the pressures associated with water and nutrient transport, some cells deposit a secondary cell wall, a rigid matrix of polysaccharide and phenolic biopolymers. The biosynthesis and deposition of these materials and the patterning of secondary wall-forming cells is controlled by a network of transcription factors. However, recent work suggests that this network forms the core of a more complex, multilevel regulatory system. This expanded system includes epigenetic, post-transcriptional, and post-translational regulation, and is coordinated with other pathways controlling primary growth and responses to environmental stimuli. New findings expand the set of transcription factors identified as secondary cell wall regulators and reveal novel regulatory processes that further govern secondary wall biogenesis.

Rice Genome-Scale Network Integration Reveals Transcriptional Regulators of Grass Cell Wall Synthesis.

Grasses have evolved distinct cell wall composition and patterning relative to dicotyledonous plants. However, despite the importance of this plant family, transcriptional regulation of its cell wall biosynthesis is poorly understood. To identify grass cell wall-associated transcription factors, we constructed the Rice Combined mutual Ranked Network (RCRN). The RCRN covers >90% of annotated rice (Oryza sativa) genes, is high quality, and includes most grass-specific cell wall genes, such as mixed-linkage glucan synthases and hydroxycinnamoyl acyltransferases. Comparing the RCRN and an equivalent Arabidopsis network suggests that grass orthologs of most genetically verified eudicot cell wall regulators also control this process in grasses, but some transcription factors vary significantly in network connectivity between these divergent species. Reverse genetics, yeast-one-hybrid, and protoplast-based assays reveal that OsMYB61a activates a grass-specific acyltransferase promoter, which confirms network predictions and supports grass-specific cell wall synthesis genes being incorporated into conserved regulatory circuits. In addition, 10 of 15 tested transcription factors, including six novel Wall-Associated regulators (WAP1, WACH1, WAHL1, WADH1, OsMYB13a, and OsMYB13b), alter abundance of cell wall-related transcripts when transiently expressed. The results highlight the quality of the RCRN for examining rice biology, provide insight into the evolution of cell wall regulation, and identify network nodes and edges that are possible leads for improving cell wall composition.