Hierarchical Gene Regulatory Network Research Articles

Time-course swRNA-seq uncovers a hierarchical gene regulatory network in controlling the response-repair-remodeling after wounding

Wounding initiates intricate responses crucial for tissue repair and regeneration. Yet, the gene regulatory networks governing wound healing remain poorly understood. Here, employing single-worm RNA sequencing (swRNA-seq) across 12 time-points, we delineated a three-stage wound repair process in C. elegans: response, repair, and remodeling. Integrating diverse datasets, we constructed a dynamic regulatory network comprising 241 transcription regulators and their inferred targets. We identified potentially seven autoregulatory TFs and five cross-autoregulatory loops involving pqm-1 and jun-1. We revealed that TFs might interact with chromatin factors and form TF-TF combinatory modules via intrinsically disordered regions to enhance response robustness. We experimentally validated six regulators functioning in transcriptional and translocation-dependent manners. Notably, nhr-76, daf-16, nhr-84, and oef-1 are potentially required for efficient repair, while elt-2 may act as an inhibitor. These findings elucidate transcriptional responses and hierarchical regulatory networks during C. elegans wound repair, shedding light on mechanisms underlying tissue repair and regeneration.

The key pathways for drought tolerance in Cerasus humilis were unveiled through transcriptome analysis.

Drought stress exerts a significant impact on the growth, development, and yield of fruit trees. Cerasus humilis is an endemic drought-resistant fruit tree in northern China. To elucidate the underlying mechanism of drought resistance in C. humilis, comprehensive physiological measurements and transcriptome analysis were conducted on the leaves of C. humilis subjected to 15- or 22-days of drought stress. We identified multiple GO terms and KEGG pathways associated with the drought stress response by performing GO and KEGG analysis on DEGs. Furthermore, through the prediction of transcription factors (TFs) and analysis of their expression levels, we observed differential expression patterns among most members of stress-responsive TF families as the duration of drought stress increased. WGCNA analysis was performed on the transcriptome to identify gene cluster modules that exhibited a strong correlation with the durations of drought. Subsequently, these modules underwent GO and KEGG enrichment analyses. The study revealed that the TF-mediated lignin biosynthesis pathway, along with the plant hormone signal transduction pathway, played a prominent role in responding to drought stress of C. humilis. Gene profiling analysis, qRT-PCR, and determination of phytohormone and lignin contents further supported this hypothesis. The hierarchical gene regulatory network was finally constructed based on DEGs from the aforementioned key enriched pathways to predict the gene regulatory mechanisms in response to stress for C. humilis. The findings from this study provide valuable insights into how C. humilis copes with drought stress while analyzing crucial gene pathways associated with its resistance from a TF perspective. This research is significant for the genetic breeding of economic forests.

The transcriptional landscape of Populus pattern/effector-triggered immunity and how PagWRKY18 involved in it.

Plants trigger a robust immune response by activating massive transcriptome reprogramming through crosstalk between PTI and ETI. However, how PTI and ETI contribute to the quantitative or/and qualitative output of immunity and how they work together when both are being activated were unclear. In this study, we performed a comprehensive overview of pathogen-triggered transcriptomic reprogramming by analyzing temporal changes in the transcriptome up to 144 h after Colletotrichum gloeosporioides inoculated in Populus. Moreover, we constructed a hierarchical gene regulatory network of PagWRKY18 and its potential target genes to explore the underlying regulatory mechanisms of PagWRKY18 that are not yet clear. Interestingly, we confirmed that PagWRKY18 protein can directly bind the W-box elements in the promoter of a transmembrane leucine-rich repeat receptor-like kinase, PagSOBIR1 gene, to trigger PTI. At the same time, PagWRKY18 functions in disease tolerance by modulation of ROS homeostasis and induction of cell death via directly targeting PagGSTU7 and PagPR4 respectively. Furthermore, PagPR4 can interact with PagWRKY18 to inhibit the expression of PagPR4 genes, forming a negative feedback loop. Taken together, these results suggest that PagWRKY18 may be involved in regulating crosstalk between PTI and ETI to activate a robust immune response and maintain intracellular homeostasis.

PagSOD2a improves poplar salt tolerance by elevating superoxide dismutase activity and decreasing malondialdehyde contents.

Superoxide dismutase (SOD) is widely present in plants and plays a crucial role in defending against oxidative stress and preventing tissue damage. This study discovered that the PagSOD2a gene in 84K poplar (Populus alba × P. glandulosa) exhibits a distinct capacity to be induced in response to salt stress. To delve into the pivotal role of PagSOD2a in conferring salt tolerance, the entire PagSOD2a fragment was successfully cloned from 84K poplar and the potential function of PagSOD2a was explored using bioinformatics and subcellular localization. PagSOD2a was found to encode a CuZn-SOD protein localized in chloroplasts. Furthermore, six CuZn-SOD family members were identified in poplar, with closely related members displaying similar gene structures, indicating evolutionary conservation. Morphological and physiological indexes of transgenic 84K poplar overexpressing PagSOD2a (OE) were compared with non-transgenic wild-type (WT) plants under salt stress. The OE lines (OE1 and OE3) showed improved growth performance, characterized by increased plant height and fresh weight, along with reduced malondialdehyde (MDA) content and electrolyte leakage rate under salt stress. Meanwhile, overexpression of PagSOD2a significantly augmented CuZn-SOD and total SOD enzyme activities, leading to a reduction in superoxide anion accumulation and an enhancement of salt tolerance. Additionally, co-expression and multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PagSOD2a constructed using transcriptome data revealed that PagSOD2a gene may be directly regulated by SPL13, NGA1b and FRS5, as well as indirectly regulated by MYB102 and WRKY6, in response to salt stress. These findings provide a theoretical and material foundation for further elucidating the function of PagSOD2a under salt stress and for developing salt-tolerant poplar varieties.

Deciphering the intricate hierarchical gene regulatory network: unraveling multi-level regulation and modifications driving secondary cell wall formation.

Wood quality is predominantly determined by the amount and the composition of secondary cell walls (SCWs). Consequently, unraveling the molecular regulatory mechanisms governing SCW formation is of paramount importance for genetic engineering aimed at enhancing wood properties. Although SCW formation is known to be governed by a hierarchical gene regulatory network (HGRN), our understanding of how a HGRN operates and regulates the formation of heterogeneous SCWs for plant development and adaption to ever-changing environment remains limited. In this review, we examined the HGRNs governing SCW formation and highlighted the significant key differences between herbaceous Arabidopsis and woody plant poplar. We clarified many confusions in existing literatures regarding the HGRNs and their orthologous gene names and functions. Additionally, we revealed many network motifs including feed-forward loops, feed-back loops, and negative and positive autoregulation in the HGRNs. We also conducted a thorough review of post-transcriptional and post-translational aspects, protein-protein interactions, and epigenetic modifications of the HGRNs. Furthermore, we summarized how the HGRNs respond to environmental factors and cues, influencing SCW biosynthesis through regulatory cascades, including many regulatory chains, wiring regulations, and network motifs. Finally, we highlighted the future research directions for gaining a further understanding of molecular regulatory mechanisms underlying SCW formation.

Comprehensive physiological, cytological, and transcriptional regulatory analyses reveal the coloration mechanism of culm yellow-slot mutation in moso bamboo (Phyllostachys edulis)

The green tissue in the culm of bamboo, engaging in photosynthesis, supplies carbohydrates to support the rapid growth of its shoots, yet the molecular basis for the chlorosis of bamboo culm tissue has not been elucidated. In contrast to the wild-type moso bamboo (Phyllostachys edulis f. pubescens, MB) with integral-green internodes at culms, the mutant one (P. edulis f. luteosulcata, YM) has typical bicolor internodes with yellow slot and green wall. By pigment quantification and chloroplast ultrastructure investigation, we found that the chlorosis in YM slot was caused by chlorophyll a deficiency, defective stroma thylakoids and misalignment of grana thylakoids. Based on transcriptomic comparisons across yellow and green tissues, the expression of plentiful genes involved in chloroplast components were significantly deceased in the tissue of YM slot, including genes associated with chloroplast development and chlorophyll metabolism. Analysis of differentially expressed genes and a multilayered hierarchical gene regulatory network both suggest that the decreased expression of BBX15s (B-BOX ZINC FINGER PROTEIN 15s) is likely the underlying cause of thylakoid dysgenesis and chlorophyll deficiency in the YM slot. This was further corroborated by Yeast one-hybrid (Y1H) assays and transgenic validation experiments, which demonstrated that BBX15s can directly regulate the transcription of genes related to chloroplast development and chlorophyll synthesis. This study provides insight into the mechanism of moso bamboo culm chloroplast development and serves as a productive basis for further exploration of bamboo culm coloration.

Conserved hierarchical gene regulatory networks for drought and cold stress response in Myrica rubra.

Stress response in plant is regulated by a large number of genes co-operating in diverse networks that serve multiple adaptive process. To understand how gene regulatory networks (GRNs) modulating abiotic stress responses, we compare the GRNs underlying drought and cold stresses using samples collected at 4 or 6h intervals within 48h in Chinese bayberry (Myrica rubra). We detected 7,583 and 8,840 differentially expressed genes (DEGs) under drought and cold stress respectively, which might be responsive to environmental stresses. Drought- and cold-responsive GRNs, which have been built according to the timing of transcription under both abiotic stresses, have a conserved trans-regulator and a common regulatory network. In both GRNs, basic helix-loop-helix family transcription factor (bHLH) serve as central nodes. MrbHLHp10 transcripts exhibited continuous increase in the two abiotic stresses and acts upstream regulator of ASCORBATE PEROXIDASE (APX) gene. To examine the potential biological functions of MrbHLH10, we generated a transgenic Arabidopsis plant that constitutively overexpresses the MrbHLH10 gene. Compared to wild-type (WT) plants, overexpressing transgenic Arabidopsis plants maintained higher APX activity and biomass accumulation under drought and cold stress. Consistently, RNAi plants had elevated susceptibility to both stresses. Taken together, these results suggested that MrbHLH10 mitigates abiotic stresses through the modulation of ROS scavenging.

ResVAE ensemble: Unsupervised identification of gene sets in multi-modal single-cell sequencing data using deep ensembles.

Feature identification and manual inspection is currently still an integral part of biological data analysis in single-cell sequencing. Features such as expressed genes and open chromatin status are selectively studied in specific contexts, cell states or experimental conditions. While conventional analysis methods construct a relatively static view on gene candidates, artificial neural networks have been used to model their interactions after hierarchical gene regulatory networks. However, it is challenging to identify consistent features in this modeling process due to the inherently stochastic nature of these methods. Therefore, we propose using ensembles of autoencoders and subsequent rank aggregation to extract consensus features in a less biased manner. Here, we performed sequencing data analyses of different modalities either independently or simultaneously as well as with other analysis tools. Our resVAE ensemble method can successfully complement and find additional unbiased biological insights with minimal data processing or feature selection steps while giving a measurement of confidence, especially for models using stochastic or approximation algorithms. In addition, our method can also work with overlapping clustering identity assignment suitable for transitionary cell types or cell fates in comparison to most conventional tools.

The multilayered hierarchical gene regulatory network reveals interaction of transcription factors in response to cadmium in Tamarix hispida roots.

Cadmium (Cd) is a toxic metal that affects the normal growth and development of plants. Roots may directly contact Cd and thus serve as the first barrier in the defense responses of plants. In this study, Tamarix hispida (T. hispida) roots treated with 150μM CdCl2 were collected for RNA-seq. A total of 2004 differentially expressed genes (DEGs) were identified at different time points. Kyoto Encyclopedia of Genes and Genomes enrichment revealed that the DEGs were significantly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis and other metabolic pathways. To explore the regulatory role of transcription factors (TFs) involved in the Cd stress response, a multilayer hierarchical gene regulatory network (ML-hGRN) was constructed, including 53 TFs and 54 structural genes in ML-hGRN, with 341 predicted regulatory relationships. Binding of DRE1A, MYC1, FEZ, ERF4 and ERF17 to predicted target genes was detected by ChIP-PCR, and DRE1A, MYC1 and FEZ were transiently overexpressed in T. hispida. The results suggest that these TFs play a key role in the Cd stress response by scavenging reactive oxygen species. In conclusion, this study predicts some Cd-responsive TFs that may have an important function under Cd stress and provides useful information for molecular breeding.

Construction of a Hierarchical Gene Regulatory Network to Reveal the Drought Tolerance Mechanism of Shanxin Poplar

Drought stress is a common adverse environment that plants encounter, and many drought-tolerant genes have been characterized. The gene regulatory network (GRN) is important in revealing the drought tolerance mechanism. Here, to investigate the regulatory mechanism of Shanxin poplar (Populus davidiana × P. bolleana) responding to drought stress, a three-layered GRN was built, and the regulatory relationship between genes in the GRN were predicted from expression correlation using a partial correlation coefficient-based algorithm. The GRN contains 1869 regulatory relationships, and includes 11 and 19 transcription factors (TFs) in the first and second layers, respectively, and 158 structural genes in the bottom layers involved in eight enriched biological processes. ChIP-PCR and qRT-PCR based on transient transformation were performed to validate the reliability of the GRN. About 88.0% of predicted interactions between the first and second layers, and 82.0% of predicted interactions between the second and third layers were correct, suggesting that the GRN is reliable. Six TFs were randomly selected from the top layer for characterizing their function in drought, and all of these TFs can confer drought tolerance. The important biological processes related to drought tolerance were identified, including "response to jasmonic acid", "response to oxidative stress", and "response to osmotic stress". In this GRN, PdbERF3 is predicted to play an important role in drought tolerance. Our data revealed the key regulators, TF-DNA interactions, and the main biological processes involved in adaption of drought stress in Shanxin poplar.

Towards a hierarchical gene regulatory network underlying somatic embryogenesis.

Genome-editing technologies have advanced in recent years but designing future crops remains limited by current methods of improving somatic embryogenesis (SE) capacity. In this Opinion, we provide an update on the molecular event by which the phytohormone auxin promotes the acquisition of plant cell totipotency through evoking massive changes in transcriptome and chromatin accessibility. We propose that the chromatin states and individual totipotency-related transcription factors (TFs) from disparate gene families organize into a hierarchical gene regulatory network underlying SE. We conclude with a discussion of the practical paths to probe the cellular origin of the somatic embryo and the epigenetic landscape of the totipotent cell state in the era of single-cell genomics.

Two high hierarchical regulators, PuMYB40 and PuWRKY75, control the low phosphorus driven adventitious root formation in Populus ussuriensis.

SummaryAdventitious rooting is an essential biological process in the vegetative propagation of economically important horticultural and forest tree species. It enables utilization of the elite genotypes in breeding programmes and production. Promotion of adventitious root (AR) formation has been associated with starvation of inorganic phosphate and some factors involved in low phosphorus (LP) signalling. However, the regulatory mechanism underlying LP‐mediated AR formation remains largely elusive. We established an efficient experimental system that guaranteed AR formation through short‐term LP treatment in Populus ussuriensis. We then generated a time‐course RNA‐seq data set to recognize key regulatory genes and regulatory cascades positively regulating AR formation through data analysis and gene network construction, which were followed by experimental validation and characterization. We constructed a multilayered hierarchical gene regulatory network, from which PuMYB40, a typical R2R3‐type MYB transcription factor (TF), and its interactive partner, PuWRKY75, as well as their direct targets, PuLRP1 and PuERF003, were identified to function upstream of the known adventitious rooting genes. These regulatory genes were functionally characterized and proved their roles in promoting AR formation in P. ussuriensis. In conclusion, our study unveiled a new hierarchical regulatory network that promoted AR formation in P. ussuriensis, which was activated by short‐term LP stimulus and primarily governed by PuMYB40 and PuWRKY75.

Construction of two regulatory networks related to salt stress and lignocellulosic synthesis under salt stress based on a Populus davidiana × P. bolleana transcriptome analysis.

Construction of ML-hGRN for the salt pathway in Populus davidiana × P. bolleana. Construction of ML-hGRN for the lignocellulosic pathway in Populus davidiana × P. bolleana under salt stress. Many woody plants, including Populus davidiana × P. bolleana, have made great contributions to human production and life. High salt is one of the main environmental factors that restricts the growth of poplar. This study found that high salt could induce strong biochemical changes in poplar. To detect the effect of salt treatment on gene expression, 18 libraries were sequenced on the Illumina sequencing platform. The results identified a large number of early differentially expressed genes (DEGs) and a small number of late DEGs, which indicated that most of the salt response genes of poplar were early response genes. In addition, 197 TFs, including NAC, ERF, and other TFs related to salt stress, were differentially expressed during salt treatment, which indicated that these TFs may play an important role in the salt stress response of poplar. Based on the RNA-seq analysis results, multilayered hierarchical gene regulatory networks (ML-hGRNs) of salt stress- and lignocellulosic synthesis-related DEGs were constructed using the GGM algorithm. The lignocellulosic synthesis regulatory network under salt stress revealed that lignocellulosic synthesis might play an important role in the process of salt stress resistance. Furthermore, the NAC family transcription factor PdbNAC83, which was found in the upper layer in both pathways, was selected to verify the accuracy of the ML-hGRNs. DAP-seq showed that the binding site of PdbNAC83 included a "TT(G/A)C(G/T)T" motif, and ChIP-PCR further verified that PdbNAC83 can regulate the promoters of at leastsix predicted downstream genes (PdbNLP2-2, PdbZFP6, PdbMYB73, PdbC2H2-like, PdbMYB93-1, PdbbHLH094) by binding to the "TT(G/A)C(G/T)T" motif, which indicates that the predicted regulatory network diagram obtained in this study is relatively accurate. In conclusion, a species-specific salt response pathway might exist in poplar, and this finding lays a foundation for further study of the regulatory mechanism of the salt stress response and provides new clues for the use of genetic engineering methods to create high-quality and highly resistant forest germplasms.

Single-cell profiling of transcriptome and histone modifications with EpiDamID

SummaryRecent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, but the combined detection of histone post-translational modifications and transcription at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins. Using these, we render the DamID technology compatible with the genome-wide identification of histone post-translational modifications. Importantly, this includes the possibility to jointly measure chromatin marks and transcription at the single-cell level. We use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. In addition, we map H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specific to notochord. Overall, EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes.

Widespread cis- and trans-regulatory evolution underlies the origin, diversification, and loss of a sexually dimorphic fruit fly pigmentation trait.

Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait-building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis-regulatory elements (CREs) that drive their expression (cis-evolution) or within genes for CRE-interacting transcription factors (trans-evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. We investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. We show these CREs to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male-specific pigmentation. We evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. We show that the transgene host frequently determines CRE activity, implicating trans-evolution as a significant factor for this trait's diversity. We validated the gain of dimorphic Bab transcription factor expression as a trans-change contributing to the dimorphic trait. Our findings suggest an amenability to change for the landscape of trans-regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs.

Evolutionarily conserved hierarchical gene regulatory networks for plant salt stress response.

Plant cells constantly alter their gene expression profiles to respond to environmental fluctuations. These continuous adjustments are regulated by multi-hierarchical networks of transcription factors. To understand how such gene regulatory networks (GRNs) have stabilized evolutionarily while allowing for species-specific responses, we compare the GRNs underlying salt response in the early-diverging and late-diverging plants Marchantia polymorpha and Arabidopsis thaliana. Salt-responsive GRNs, constructed on the basis of the temporal transcriptional patterns in the two species, share common trans-regulators but exhibit an evolutionary divergence in cis-regulatory sequences and in the overall network sizes. In both species, WRKY-family transcription factors and their feedback loops serve as central nodes in salt-responsive GRNs. The divergent cis-regulatory sequences of WRKY-target genes are probably associated with the expansion in network size, linking salt stress to tissue-specific developmental and physiological responses. The WRKY modules and highly linked WRKY feedback loops have been preserved widely in other plants, including rice, while keeping their binding-motif sequences mutable. Together, the conserved trans-regulators and the quickly evolving cis-regulatory sequences allow salt-responsive GRNs to adapt over a long evolutionary timescale while maintaining some consistent regulatory structure. This strategy may benefit plants as they adapt to changing environments.

Genetic control of retinal ganglion cell genesis

Retinal ganglion cells (RGCs) are the only projection neurons in the neural retina. They receive and integrate visual signals from upstream retinal neurons in the visual circuitry and transmit them to the brain. The function of RGCs is performed by the approximately 40 RGC types projecting to various central brain targets. RGCs are the first cell type to form during retinogenesis. The specification and differentiation of the RGC lineage is a stepwise process; a hierarchical gene regulatory network controlling the RGC lineage has been identified and continues to be elaborated. Recent studies with single-cell transcriptomics have led to unprecedented new insights into their types and developmental trajectory. In this review, we summarize our current understanding of the functions and relationships of the many regulators of the specification and differentiation of the RGC lineage. We emphasize the roles of these key transcription factors and pathways in different developmental steps, including the transition from retinal progenitor cells (RPCs) to RGCs, RGC differentiation, generation of diverse RGC types, and central projection of the RGC axons. We discuss critical issues that remain to be addressed for a comprehensive understanding of these different aspects of RGC genesis and emerging technologies, including single-cell techniques, novel genetic tools and resources, and high-throughput genome editing and screening assays, which can be leveraged in future studies.

A systems biology approach identifies a regulator, <i>BplERF1</i>, of cold tolerance in <i>Betula platyphylla</i>

Cold is an abiotic stress that can greatly affect the growth and survival of plants. Here, we reported that an <i>AP2/ERF</i> family gene, <i>BplERF1</i>, isolated from <i>Betula platyphylla</i> played a contributing role in cold stress tolerance. Overexpression of <i>BplERF1</i> in <i>B. platyphylla</i> transgenic lines enhanced cold stress tolerance by increasing the scavenging capability and reducing H₂O₂ and malondialdehyde (MDA) content in transgenic plants. Construction of BplERF-mediated multilayered hierarchical gene regulatory network (ML-hGRN), using Top-down GGM algorithm and the transcriptomic data of <i>BplERF1</i> overexpression lines, led to the identification of five candidate target genes of BplERF1 which include <i>MPK20</i>, <i>ERF9</i>, <i>WRKY53</i>, <i>WRKY70</i>, and <i>GIA1</i>. All of them were then verified to be the true target genes of BplERF1 by chromatin-immunoprecipitation PCR (ChIP-PCR) assay. Our results indicate that BplERF1 is a positive regulator of cold tolerance and is capable of exerting regulation on the expression of cold signaling and regulatory genes, causing mitigation of reactive oxygen species.

Chromatin Accessibility Dynamics and a Hierarchical Transcriptional Regulatory Network Structure for Plant Somatic Embryogenesis.

Plant somatic embryogenesis refers to a phenomenon where embryos develop from somatic cells in the absence of fertilization. Previous studies have revealed that the phytohormone auxin plays a crucial role in somatic embryogenesis by inducing a cell totipotent state, although its underlying mechanism is poorly understood. Here, we show that auxin rapidly rewires the cell totipotency network by altering chromatin accessibility. The analysis of chromatin accessibility dynamics further reveals a hierarchical gene regulatory network underlying somatic embryogenesis. Particularly, we find that the embryonic nature of explants is a prerequisite for somatic cell reprogramming. Upon cell reprogramming, the B3-type totipotent transcription factor LEC2 promotes somatic embryo formation by direct activation of the early embryonic patterning genes WOX2 and WOX3. Our results thus shed light on the molecular mechanism by which auxin promotes the acquisition of plant cell totipotency and establish a direct link between cell totipotent genes and the embryonic development pathway.

PuHox52-mediated hierarchical multilayered gene regulatory network promotes adventitious root formation in Populus ussuriensis.

Adventitious root (AR) formation is critically important in vegetative propagation through cuttings in some plants, especially woody species. However, the underlying molecular mechanisms remain elusive. Here, we report the identification of a poplar homeobox gene, PuHox52, which was induced rapidly (within 15min) at the basal ends of stems upon cutting and played a key regulatory role in adventitious rooting. We demonstrated that overexpression of PuHox52 significantly increased the number of ARs while suppression of PuHox52 had the opposite effect. A multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PuHox52 was reverse-engineered and demonstrated to govern AR formation. PuHox52 regulated AR formation through upregulation of nine hub regulators, including a jasmonate signaling pathway gene, PuMYC2, and an auxin signaling pathway gene, PuAGL12. We also identified coherent type 4 feed-forward loops within this ML-hGRN; PuHox52 repressed PuHDA9, which encodes a histone deacetylase, and led to an increase in acetylation and presumably expression of three hub regulators, PuWRKY51, PuLBD21 and PuIAA7. Our results indicate that the ML-hGRN mediated by PuHox52 governs AR formation at the basal ends of stem cuttings from poplar trees.