Disruption of p-coumaroyl-CoA:monolignol transferases in rice drastically alters lignin composition.

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants' cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice.

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (-14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance.

Multifunctional 5-hydroxyconiferaldehyde O-methyltransferases (CAldOMTs) in plant metabolism.

Lignin, flavonoids, melatonin, and stilbenes are plant specialized metabolites with diverse physiological and biological functions, supporting plant growth and conferring stress resistance. Their biosynthesis requires O-methylations catalyzed by 5-hydroxyconiferaldehyde O-methyltransferase (CAldOMT; also called caffeic acid O-methyltransferase, COMT). CAldOMT was first known for its roles in syringyl (S) lignin biosynthesis in angiosperm cell walls and later found to be multifunctional. This enzyme also catalyzes O-methylations in flavonoid, melatonin, and stilbene biosynthetic pathways. Phylogenetic analysis indicated the convergent evolution of enzymes with OMT activities towards the monolignol biosynthetic pathway intermediates in some gymnosperm species that lack S-lignin and Selaginella moellendorffii, a lycophyte which produces S-lignin. Furthermore, neofunctionalization of CAldOMTs occurred repeatedly during evolution, generating unique O-methyltransferases (OMTs) with novel catalytic activities and/or accepting novel substrates, including lignans, 1,2,3-trihydroxybenzene, and phenylpropenes. This review summarizes multiple aspects of CAldOMTs and their related proteins in plant metabolism and discusses their evolution, molecular mechanism, and roles in biorefineries, agriculture, and synthetic biology.

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.

Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

Overexpression of a rice BAHD acyltransferase gene in switchgrass (Panicum virgatum L.) enhances saccharification.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a promising bioenergy feedstock because it can be grown on marginal land and produces abundant biomass. Recalcitrance of the lignocellulosic components of the switchgrass cell wall to enzymatic degradation into simple sugars impedes efficient biofuel production. We previously demonstrated that overexpression of OsAT10, a BAHD acyltransferase gene, enhances saccharification efficiency in rice. RESULTS: Here we show that overexpression of the rice OsAT10 gene in switchgrass decreased the levels of cell wall-bound ferulic acid (FA) in green leaf tissues and to a lesser extent in senesced tissues, and significantly increased levels of cell wall-bound p-coumaric acid (p-CA) in green leaves but decreased its level in senesced tissues of the T0 plants under greenhouse conditions. The engineered switchgrass lines exhibit an approximate 40% increase in saccharification efficiency in green tissues and a 30% increase in senesced tissues. CONCLUSION: Our study demonstrates that overexpression of OsAT10, a rice BAHD acyltransferase gene, enhances saccharification of lignocellulosic biomass in switchgrass.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development.

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

Development of an integrated transcript sequence database and a gene expression atlas for gene discovery and analysis in switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.) is a perennial C4 grass with the potential to become a major bioenergy crop. To help realize this potential, a set of RNA-based resources were developed. Expressed sequence tags (ESTs) were generated from two tetraploid switchgrass genotypes, Alamo AP13 and Summer VS16. Over 11.5 million high-quality ESTs were generated with 454 sequencing technology, and an additional 169 079 Sanger sequences were obtained from the 5' and 3' ends of 93 312 clones from normalized, full-length-enriched cDNA libraries. AP13 and VS16 ESTs were assembled into 77 854 and 30 524 unique transcripts (unitranscripts), respectively, using the Newbler and pave programs. Published Sanger-ESTs (544 225) from Alamo, Kanlow, and 15 other cultivars were integrated with the AP13 and VS16 assemblies to create a universal switchgrass gene index (PviUT1.2) with 128 058 unitranscripts, which were annotated for function. An Affymetrix cDNA microarray chip (Pvi_cDNAa520831) containing 122 973 probe sets was designed from PviUT1.2 sequences, and used to develop a Gene Expression Atlas for switchgrass (PviGEA). The PviGEA contains quantitative transcript data for all major organ systems of switchgrass throughout development. We developed a web server that enables flexible, multifaceted analyses of PviGEA transcript data. The PviGEA was used to identify representatives of all known genes in the phenylpropanoid-monolignol biosynthesis pathway.

Comparative genomic analysis of the R2R3 MYB secondary cell wall regulators of Arabidopsis, poplar, rice, maize, and switchgrass.

BACKGROUND: R2R3 MYB proteins constitute one of the largest plant transcription factor clades and regulate diverse plant-specific processes. Several R2R3 MYB proteins act as regulators of secondary cell wall (SCW) biosynthesis in Arabidopsis thaliana (At), a dicotyledenous plant. Relatively few studies have examined SCW R2R3 MYB function in grasses, which may have diverged from dicots in terms of SCW regulatory mechanisms, as they have in cell wall composition and patterning. Understanding cell wall regulation is especially important for improving lignocellulosic bioenergy crops, such as switchgrass. RESULTS: Here, we describe the results of applying phylogenic, OrthoMCL, and sequence identity analyses to classify the R2R3 MYB family proteins from the annotated proteomes of Arabidposis, poplar, rice, maize and the initial genome (v0.0) and translated transcriptome of switchgrass (Panicum virgatum). We find that the R2R3 MYB proteins of the five species fall into 48 subgroups, including three dicot-specific, six grass-specific, and two panicoid grass-expanded subgroups. We observe four classes of phylogenetic relationships within the subgroups of known SCW-regulating MYB proteins between Arabidopsis and rice, ranging from likely one-to-one orthology (for AtMYB26, AtMYB103, AtMYB69) to no homologs identifiable (for AtMYB75). Microarray data for putative switchgrass SCW MYBs indicate that many maintain similar expression patterns with the Arabidopsis SCW regulators. However, some of the switchgrass-expanded candidate SCW MYBs exhibit differences in gene expression patterns among paralogs, consistent with subfunctionalization. Furthermore, some switchgrass representatives of grass-expanded clades have gene expression patterns consistent with regulating SCW development. CONCLUSIONS: Our analysis suggests that no single comparative genomics tool is able to provide a complete picture of the R2R3 MYB protein family without leaving ambiguities, and establishing likely false-negative and -positive relationships, but that used together a relatively clear view emerges. Generally, we find that most R2R3 MYBs that regulate SCW in Arabidopsis are likely conserved in the grasses. This comparative analysis of the R2R3 MYB family will facilitate transfer of understanding of regulatory mechanisms among species and enable control of SCW biosynthesis in switchgrass toward improving its biomass quality.

Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification.

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.

Towards establishment of a rice stress response interactome.

Rice (Oryza sativa) is a staple food for more than half the world and a model for studies of monocotyledonous species, which include cereal crops and candidate bioenergy grasses. A major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%-60% yield losses globally each year. To elucidate stress response signaling networks, we constructed an interactome of 100 proteins by yeast two-hybrid (Y2H) assays around key regulators of the rice biotic and abiotic stress responses. We validated the interactome using protein-protein interaction (PPI) assays, co-expression of transcripts, and phenotypic analyses. Using this interactome-guided prediction and phenotype validation, we identified ten novel regulators of stress tolerance, including two from protein classes not previously known to function in stress responses. Several lines of evidence support cross-talk between biotic and abiotic stress responses. The combination of focused interactome and systems analyses described here represents significant progress toward elucidating the molecular basis of traits of agronomic importance.

Grass lignin: biosynthesis, biological roles, and industrial applications.

Lignin is a phenolic heteropolymer found in most terrestrial plants that contributes an essential role in plant growth, abiotic stress tolerance, and biotic stress resistance. Recent research in grass lignin biosynthesis has found differences compared to dicots such as Arabidopsis thaliana. For example, the prolific incorporation of hydroxycinnamic acids into grass secondary cell walls improve the structural integrity of vascular and structural elements via covalent crosslinking. Conversely, fundamental monolignol chemistry conserves the mechanisms of monolignol translocation and polymerization across the plant phylum. Emerging evidence suggests grass lignin compositions contribute to abiotic stress tolerance, and periods of biotic stress often alter cereal lignin compositions to hinder pathogenesis. This same recalcitrance also inhibits industrial valorization of plant biomass, making lignin alterations and reductions a prolific field of research. This review presents an update of grass lignin biosynthesis, translocation, and polymerization, highlights how lignified grass cell walls contribute to plant development and stress responses, and briefly addresses genetic engineering strategies that may benefit industrial applications.

Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase-Client (KiC) assay.

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or "clients", remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1's substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

Enhancing monolignol ferulate conjugate levels in poplar lignin via OsFMT1.

BACKGROUND: The phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass. RESULTS: Overexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV-Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic p oplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars. CONCLUSIONS: The findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass.

The rapid-tome, a 3D-printed microtome, and an updated hand-sectioning method for high-quality plant sectioning.

BACKGROUND: Microscopic analysis of plant anatomy is a common procedure in biology to study structure and function that requires high-quality sections for accurate measurements. Hand sectioning of specimens is typically limited to moderately soft tissue while harder samples prohibit sectioning by hand and/or result in inconsistent thicknesses. RESULTS: Here we present both a clearly described hand-sectioning method and a novel microtome design that together provide the means to section a variety of plant sample types. The described hand-sectioning method for herbaceous stems works well for softer subjects but is less suitable for samples with secondary growth (e.g., wood production). Instead, the "Rapid-Tome" is a novel tool for sectioning both soft and tougher high-aspect-ratio samples, such as stems and roots, with excellent sample control. The Rapid-Tome can be 3D-printed in approximately 18 h on a mid-quality printer common at university maker spaces. After printing and trimming, Rapid-Tome assembly takes a few minutes with five metal parts common at hardware stores. Users sectioned a variety of plant samples including the hollow internodes of switchgrass (Panicum virgatum), fibrous switchgrass roots containing aerenchyma, and woody branches of eastern red cedar (Juniperus virginiana) and American sycamore (Platanus occidentalis). A comparative analyses with Rapid-Tome-produced sections readily revealed a significant difference in seasonal growth of sycamore xylem vessel area in spring (49%) vs. summer (23%). Additionally, high school students with no prior experience produced sections with the Rapid-Tome adequate for comparative analyses of various plant samples in less than an hour. CONCLUSIONS: The described hand-sectioning method is suitable for softer tissues, including hollow-stemmed grasses and similar samples. In addition, the Rapid-Tome provides capacity to safely produce high-quality sections of tougher plant materials at a fraction of the cost of traditional microtomes combined with excellent sample control. The Rapid-Tome features rapid sectioning, sample advancement, blade changes, and sample changes; it is highly portable and can be used easily with minimal training making production of thin sections accessible for classroom and outreach use, in addition to research.

Corrigendum: Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

[This corrects the article DOI: 10.3389/fpls.2022.1088879.].

Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

In the last decade it has become clear that enzymes in the "BAHD" family of acyl-CoA transferases play important roles in the addition of phenolic acids to form ester-linked moieties on cell wall polymers. We focus here on the addition of two such phenolics-the hydroxycinnamates, ferulate and p-coumarate-to two cell wall polymers, glucuronoarabinoxylan and to lignin. The resulting ester-linked feruloyl and p-coumaroyl moities are key features of the cell walls of grasses and other commelinid monocots. The capacity of ferulate to participate in radical oxidative coupling means that its addition to glucuronoarabinoxylan or to lignin has profound implications for the properties of the cell wall - allowing respectively oxidative crosslinking to glucuronoarabinoxylan chains or introducing ester bonds into lignin polymers. A subclade of ~10 BAHD genes in grasses is now known to (1) contain genes strongly implicated in addition of p-coumarate or ferulate to glucuronoarabinoxylan (2) encode enzymes that add p-coumarate or ferulate to lignin precursors. Here, we review the evidence for functions of these genes and the biotechnological applications of manipulating them, discuss our understanding of mechanisms involved, and highlight outstanding questions for future research.

Plant Biology: New Insight into How Roots 'Mask Up'.

Extra- and trans-cellular barriers are essential for root function under even mild stress. New research shows that establishing both the lignin and suberin barriers in the Arabidopsis endodermis requires phenylpropanoid biosynthesis by endodermal cells themselves.

Phenylpropanoid Biosynthesis Gene Expression Precedes Lignin Accumulation During Shoot Development in Lowland and Upland Switchgrass Genotypes.

Efficient conversion of lignocellulosic biomass into biofuels is influenced by biomass composition and structure. Lignin and other cell wall phenylpropanoids, such as para-coumaric acid (pCA) and ferulic acid (FA), reduce cell wall sugar accessibility and hamper biochemical fuel production. Toward identifying the timing and key parameters of cell wall recalcitrance across different switchgrass genotypes, this study measured cell wall composition and lignin biosynthesis gene expression in three switchgrass genotypes, A4 and AP13, representing the lowland ecotype, and VS16, representing the upland ecotype, at three developmental stages [Vegetative 3 (V3), Elongation 4 (E4), and Reproductive 3 (R3)] and three segments (S1-S3) of the E4 stage under greenhouse conditions. A decrease in cell wall digestibility and an increase in phenylpropanoids occur across development. Compared with AP13 and A4, VS16 has significantly less lignin and greater cell wall digestibility at the V3 and E4 stages; however, differences among genotypes diminish by the R3 stage. Gini correlation analysis across all genotypes revealed that lignin and pCA, but also pectin monosaccharide components, show the greatest negative correlations with digestibility. Lignin and pCA accumulation is delayed compared with expression of phenylpropanoid biosynthesis genes, while FA accumulation coincides with expression of these genes. The different cell wall component accumulation profiles and gene expression correlations may have implications for system biology approaches to identify additional gene products with cell wall component synthesis and regulation functions.

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.