Wall Patterning Research Articles

Abstract We089: Numb Family Proteins in Epicardium Regulate Epicardial Cells Differentiation and Ventricular Patterning During Development

Background: Numb Family Proteins (NFPs), specifically two mammalian homologs Numb (denoted by Nb ) and Numblike (denoted by Nl ), have been evident to regulate essential cardiac functions during development. Previous studies reported NFPs as integral for epicardial cells (EpiCs) epithelial mesenchymal-transition (EMT). However, whether NFPs in epicardium regulate EpiCs differentiation to affect cardiac morphogenesis have not been explored. In this study, we investigated how NFPs in epicardium regulate EpiCs EMT and differentiation to modulate cardiac development and subsequently affect cardiac maintenance in adult stages. Method: Using mCherry:Numb knockin mouse model we observed enrichment of Numb in epicardium. We deleted Nb and Nl in epicardium via Tbx18 Cre/+ lineage and refer to this as EDKO. We harvested hearts at embryonic day 15.5/18.5 and assessed morphological and mechanistic differences between control and EDKOs. Conventional and Speckle tracking Echocardiography were used to examine adult heart structure and functions. Results: Our findings revealed that EDKO embryonic hearts display specific morphology with extended left ventricles. We found a surprising difference between female and male EDKOs in terms of survival to adulthood. Reduced EpiCs EMT and Cardiac Fibroblasts (CFs) migration distances, changes in CFs distribution have been observed in EDKO developing hearts. Reduced expression of smooth muscle cells (SMCs) and pericytes also was noticed in EDKOs. Strikingly, EDKO embryonic hearts show defects in ventricular compaction, ventricular wall patterning and in cell proliferation. Furthermore, when survived to adult stage, EDKO hearts revealed significant structural and functional changes over their aging. While digging into regulatory pathways, we found changes in Fibroblast growth factor receptors (Fgfrs)/p-Erk pathway and in activation of Notch1 in EDKO hearts. Conclusion: NFPs in epicardium regulate EpiCs differentiation into CFs and SMCs, and regulate ventricular wall morphogenesis during development. NFPs are also required to maintain cardiac structure and function at adult stages. Fgfrs/p-Erk pathway and Notch1 could be the possible mechanism behind cardiac morphogenetic regulation by NFPs in epicardium. Future aims: Further studies will identify downstream targets of NFPs’ regulation of EpiCs differentiation and ventricular wall patterning in embryonic hearts, and mechanistic pathways of epicardial NFPs’ role in adult hearts.

Microtubule-associated phase separation of MIDD1 tunes cell wall spacing in xylem vessels in Arabidopsis thaliana.

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Xylan clustering on the pollen surface is required for exine patterning.

Xylan is a crosslinking polymer that plays an important role in the assembly of heterogeneous cell wall structures in plants. The pollen wall, a specialized cell wall matrix, exhibits diverse sculpted patterns that serve to protect male gametophytes and facilitate pollination during plant reproduction. However, whether xylan is precisely anchored into clusters and its influence on pollen wall patterning remain unclear. Here, we report xylan clustering on the mature pollen surface in different plant species that is indispensable for the formation of sculpted exine patterns in dicot and monocot plants. Chemical composition analyses revealed that xylan is generally present at low abundance in the mature pollen of flowering plants and shows plentiful variations in terms of substitutions and modifications. Consistent with the expression profiles of their encoding genes, genetic characterization revealed IRREGULAR XYLEM10-LIKE (IRX10L) and its homologous proteins in the GT47 family of glycosyltransferases as key players in the formation of these xylan micro-/nano-compartments on the pollen surface in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). A deficiency in xylan biosynthesis abolished exine patterning on pollen and compromised male fertility. Therefore, our study outlines a mechanism of exine patterning and provides a tool for manipulating male fertility in crop breeding.

Microtubule nucleation complex behavior is critical for cortical array homogeneity and xylem wall patterning

Plant cell walls are versatile materials that can adopt a wide range of mechanical properties through controlled deposition of cellulose fibrils. Wall integrity requires a sufficiently homogeneous fibril distribution to cope effectively with wall stresses. Additionally, specific conditions, such as the negative pressure in water transporting xylem vessels, may require more complex wall patterns, e.g., bands in protoxylem. The orientation and patterning of cellulose fibrils are guided by dynamic cortical microtubules. New microtubules are predominantly nucleated from parent microtubules causing positive feedback on local microtubule density with the potential to yield highly inhomogeneous patterns. Inhomogeneity indeed appears in all current cortical array simulations that include microtubule-based nucleation, suggesting that plant cells must possess an as-yet unknown balancing mechanism to prevent it. Here, in a combined simulation and experimental approach, we show that a limited local recruitment of nucleation complexes to microtubules can counter the positive feedback, whereas local tubulin depletion cannot. We observe that nucleation complexes preferentially appear at the plasma membrane near microtubules. By incorporating our experimental findings in stochastic simulations, we find that the spatial behavior of nucleation complexes delicately balances the positive feedback, such that differences in local microtubule dynamics-as in developing protoxylem-can quickly turn a homogeneous array into a banded one. Our results provide insight into how the plant cytoskeleton has evolved to meet diverse mechanical requirements and greatly increase the predictive power of computational cell biology studies.

Microtubule-associated ROP interactors affect microtubule dynamics and modulate cell wall patterning and root hair growth.

Rho of plant (ROP) proteins and the interactor of constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules that regulate metaxylem secondary cell wall patterning. Yet, loss-of-function mutants of ICR5 and its closest homologs have not been studied and, hence, the functions of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of Arabidopsis icr2 and icr5 single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings; however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, whereas icr2, but not icr5, mutants develop split root hairs, demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.

KOMPEITO, an Atypical Arabidopsis Rhomboid-Related Gene, Is Required for Callose Accumulation and Pollen Wall Development.

Fertilization is a key event for sexually reproducing plants. Pollen–stigma adhesion, which is the first step in male–female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a β-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen–stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.

Secondary cell wall patterning-connecting the dots, pits and helices.

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.

Physiological and genetic control of transpiration efficiency in African rice, Oryza glaberrima Steud.

Improving crop water use efficiency, the amount of carbon assimilated as biomass per unit of water used by a plant, is of major importance as water for agriculture becomes scarcer. In rice, the genetic bases of transpiration efficiency, the derivation of water use efficiency at the whole-plant scale, and its putative component trait transpiration restriction under high evaporative demand remain unknown. These traits were measured in 2019 in a panel of 147 African rice (Oryza glaberrima) genotypes known to be potential sources of tolerance genes to biotic and abiotic stresses. Our results reveal that higher transpiration efficiency is associated with transpiration restriction in African rice. Detailed measurements in a subset of highly contrasted genotypes in terms of biomass accumulation and transpiration confirmed these associations and suggested that root to shoot ratio played an important role in transpiration restriction. Genome wide association studies identified marker-trait associations for transpiration response to evaporative demand, transpiration efficiency, and its residuals, with links to genes involved in water transport and cell wall patterning. Our data suggest that root-shoot partitioning is an important component of transpiration restriction that has a positive effect on transpiration efficiency in African rice. Both traits are heritable and define targets for breeding rice with improved water use strategies.

The dynamic changes of tracheary elements in an intraspecific quinoa (Chenopodium quinoa) graft

Vascular connection is key to successful graft. Little study has been devoted to the behavior of tracheary elements (TEs), the basic component of vascular bundles, during vascular connection between scion and rootstock. Here we report the structural changes of TEs at the graft interface between two quinoa cultivars, Qaidam White-1 (QW1) and Qaidam Red-1 (QR1). Our results showed that TEs in ungrafted plants developed following an ontogenetic sequence, i.e., the annular vessel, helical vessel, scalariform vessel, reticulate vessel, and pitted vessel. However, this process was greatly accelerated in grafted plants, resulting in quick developmental transition of TE wall patterning. At the early stage of intraspecific grafting (e.g., 5 days after grafting), the membrane-like cellular patches were heavily accumulated at the graft interface but quickly retreated within 2–4 days, suggesting an early emergency response to grafting. The TE length in both scion and rootstock was significantly shorter (more than 50% on average, nTE = 747) than the ungrafted plants in the same period. These short TEs were gradually integrated into a long, continuous conduit, thereby enabling the functional vasculature at the graft union. In addition, the pit size was gradually reduced, for example, for the surface area of outer pit aperture, from 12.73 ± 3.15 to 5.40 ± 0.30 μm2, or for the surface area of inner pit aperture, from 9.34 ± 3.33 to 1.96 ± 1.04 μm2, in 18 days (npits = 2830). Taken together, the morphological changes of TEs and cellular responses to grafting in the intraspecific grafts seemed to be conservative to other homografts and heterografts, implying that these behavioral changes are highly adaptive to the scion–rootstock interaction.

Xylan-based nanocompartments orchestrate plant vessel wall patterning.

Nanoclustering of biomacromolecules allows cells to efficiently orchestrate biological processes. The plant cell wall is a highly organized polysaccharide network but is heterogeneous in chemistry and structure. However, polysaccharide-based nanocompartments remain ill-defined. Here, we identify a xylan-rich nanodomain at pit borders of xylem vessels. We show that these nanocompartments maintain distinct wall patterns by anchoring cellulosic nanofibrils at the pit borders, critically supporting vessel robustness, water transport and leaf transpiration. The nanocompartments are produced by the activity of IRREGULAR XYLEM (IRX)10 and its homologues, which we show are de novo xylan synthases. Our study hence outlines a mechanism of how xylans are synthesized, how they assemble into nanocompartments and how the nanocompartments sustain cell wall pit patterning to support efficient water transport throughout the plant body.

DCET1 Controls Male Sterility Through Callose Regulation, Exine Formation, and Tapetal Programmed Cell Death in Rice.

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1, having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1. Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding.

POLLEN STERILITY, a novel suppressor of cell division, is required for timely tapetal programmed cell death in rice.

Timely programmed cell death (PCD) of the tapetum supplying nutrients to microspores is a prerequisite for normal pollen development. Here we identified a unique mutant of rice (Oryza sativa L.), pollen sterility (post), which showed aborted pollens accompanied with extra-large husks. Due to failure of timely PCD of tapetal cells, post exhibited abnormal pollen wall patterning and defective pollen grains. By map-based cloning, we identified a causal gene, POST, encoding a novel protein which is ubiquitously localized in cells. RNA in situ hybridization showed that POST is highly detected in the tapetum and microspores at stages 8 and 9. Transcriptome analysis indicated that POST could function as an important regulator of the metabolic process involved in tapetal PCD. Compared with wild-type rice, post mutant has an increased cell number resulting from elevated expression of cell cycle associated genes in grain husks. Overexpression of POST inhibits grain size in wild type, while appropriate expression of POST in post mutant can recover the seed fertility but has little effect on the large grains, illustrating that fine-tuning of POST expression could be a potential strategy for rice yield improvement. The connection between cell division and cell death conferred by POST provides novel insights into the understanding of the tapetal PCD process.

Domain Wall Patterning and Giant Response Functions in Ferrimagnetic Spinels.

The manipulation of mesoscale domain wall phenomena has emerged as a powerful strategy for designing ferroelectric responses in functional devices, but its full potential is not yet realized in the field of magnetism. This work shows a direct connection between magnetic response functions in mechanically strained samples of Mn3O4 and MnV2O4 and stripe‐like patternings of the bulk magnetization which appear below known magnetostructural transitions. Building off previous magnetic force microscopy data, a small‐angle neutron scattering is used to show that these patterns represent distinctive magnetic phenomena which extend throughout the bulk of two separate materials, and further are controllable via applied magnetic field and mechanical stress. These results are unambiguously connected to the anomalously large magnetoelastic and magnetodielectric response functions reported for these materials, by performing susceptibility measurements on the same crystals and directly correlating local and macroscopic data.

Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

Pollen wall patterns as a model for biological self-assembly.

We are still far from being able to predict organisms' shapes purely from their genetic codes. While it is imperative to identify which encoded macromolecules contribute to a phenotype, determining how macromolecules self‐assemble independently of the genetic code may be equally crucial for understanding shape development. Pollen grains are typically single‐celled microgametophytes that have decorated walls of various shapes and patterns. The accumulation of morphological data and a comprehensive understanding of the wall development makes this system ripe for mathematical and physical modeling. Therefore, pollen walls are an excellent system for identifying both the genetic products and the physical processes that result in a huge diversity of extracellular morphologies. In this piece, I highlight the current understanding of pollen wall biology relevant for quantification studies and enumerate the modellable aspects of pollen wall patterning and specific approaches that one may take to elucidate how pollen grains build their beautifully patterned walls.

Defective Microspore Development1 is required for microspore cell integrity and pollen wall formation in rice.

Highly coordinated pollen wall patterning is essential for male reproductive development. Here, we report the identification of Defective Microspore Development1 (DMD1), which encodes a nuclear-localized protein possessing transactivation activity. DMD1 is preferentially expressed in the tapetum and microspores during post-meiotic development. Mutations in DMD1 cause a male-sterile phenotype with impaired microspore cell integrity. The mutants display abnormal callose degradation, accompanied by inhibited primexine thickening in the newly released microspores. Several genes associated with callose degradation and primexine formation are downregulated in dmd1 anthers. In addition, irregular Ubisch body morphology and discontinuous endexine occur, and the baculum is completely absent in dmd1. DMD1 interacts with Tapetum Degeneration Retardation (TDR), a basic helix-loop-helix transcription factor required for exine formation. Taken together, our results suggest that DMD1 is responsible for microspore cell integrity, primexine formation and exine pattern formation during Oryza sativa (rice) microspore development.

The temporal regulation of TEK contributes to pollen wall exine patterning.

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.

PERSISTENT TAPETAL CELL2 Is Required for Normal Tapetal Programmed Cell Death and Pollen Wall Patterning.

The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

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.