Single-cell RNA sequencing analysis of the embryogenic callus clarifies the spatiotemporal developmental trajectories of the early somatic embryo in Dimocarpus longan.

Plant embryogenic calli (ECs) can undergo somatic embryogenesis to regenerate plants. This process is mediated by regulatory factors, such as transcription factors and specifically expressed genes, but the precise molecular mechanisms underlying somatic embryogenesis at the single-cell level remain unclear. In this study, we performed high-resolution single-cell RNA sequencing analysis to determine the cellular changes in the EC of the woody plant species Dimocarpus longan (longan) and clarify the continuous cell differentiation trajectories at the transcriptome level. The highly heterogeneous cells in the EC were divided into 12 putative clusters (e.g., proliferating, meristematic, vascular, and epidermal cell clusters). We determined cluster-enriched expression marker genes and found that overexpression of the epidermal cell marker gene GDSL ESTERASE/LIPASE-1 inhibited the hydrolysis of triacylglycerol. In addition, the stability of autophagy was critical for the somatic embryogenesis of longan. The pseudo-timeline analysis elucidated the continuous cell differentiation trajectories from early embryonic cell division to vascular and epidermal cell differentiation during the somatic embryogenesis of longan. Moreover, key transcriptional regulators associated with cell fates were revealed. We found that ETHYLENE RESPONSIVE FACTOR 6 was characterized as a heat-sensitive factor that negatively regulates longan somatic embryogenesis under high-temperature stress conditions. The results of this study provide new spatiotemporal insights into cell division and differentiation during longan somatic embryogenesis at single-cell resolution.

ETHYLENE RESPONSE FACTOR6, A Central Regulator of Plant Growth in Response to Stress.

ETHYLENE RESPONSE FACTOR6 (ERF6) has emerged as a central player in stress-induced plant growth inhibition. It orchestrates complex pathways that enable plants to acclimate and thrive in challenging environments. In response to various abiotic and biotic stresses, ERF6 is promptly activated through both ethylene-dependent and -independent pathways, and contributes to enhanced stress tolerance mechanisms by activating a broad spectrum of genes at various developmental stages. Despite the crucial role of ERF6, there is currently a lack of published comprehensive insights into its function in plant growth and stress response. In this respect, based on the tight connection between ethylene and ERF6, we review the latest research findings on how ethylene regulates stress responses and the mechanisms involved. In addition, we summarize the trends and advances in ERF6-mediated plant performance under optimal and stressful conditions. Finally, we also highlight key questions and suggest potential paths to unravel the ERF6 regulon in future research.

RPW8.1 enhances the ethylene-signaling pathway to feedback-attenuate its mediated cell death and disease resistance in Arabidopsis.

The Arabidopsis RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) activates confined cell death and defense against different pathogens. However, the underlying regulatory mechanisms still remain elusive. Here, we show that RPW8.1 activates ethylene signaling that, in turn, negatively regulates RPW8.1 expression. RPW8.1 binds and stabilizes 1-aminocyclopropane-1-carboxylate oxidase 4 (ACO4), which may in part explain increased ethylene production and signaling in RPW8.1-expressing plants. In return, ACO4 and other key components of ethylene signaling negatively regulate RPW8.1-mediated cell death and disease resistance via suppressing RPW8.1 expression. Loss of function in ACO4, EIN2, EIN3 EIL1, ERF6, ERF016 or ORA59 increases RPW8.1-mediated cell death and defense response. By contrast, overexpression of EIN3 abolishes or significantly compromises RPW8.1-mediated cell death and disease resistance. Furthermore, ERF6, ERF016 and ORA59 appear to act as trans-repressors of RPW8.1, with OAR59 being able to directly bind to the RPW8.1 promoter. Taken together, our results have revealed a feedback regulatory circuit connecting RPW8.1 and the ethylene-signaling pathway, in which RPW8.1 enhances ethylene signaling, and the latter, in return, negatively regulates RPW8.1-mediated cell death and defense response via suppressing RPW8.1 expression to attenuate its defense activity.

VviERF6Ls: an expanded clade in Vitis responds transcriptionally to abiotic and biotic stresses and berry development.

BACKGROUND: VviERF6Ls are an uncharacterized gene clade in Vitis with only distant Arabidopsis orthologs. Preliminary data indicated these transcription factors may play a role in berry development and extreme abiotic stress responses. To better understand this highly duplicated, conserved clade, additional members of the clade were identified in four Vitis genotypes. A meta-data analysis was performed on publicly available microarray and RNA-Seq data (confirmed and expanded with RT-qPCR), and Vitis VviERF6L1 overexpression lines were established and characterized with phenotyping and RNA-Seq. RESULTS: A total of 18 PN40024 VviERF6Ls were identified; additional VviERF6Ls were identified in Cabernet Sauvignon, Chardonnay, and Carménère. The amino acid sequences of VviERF6Ls were found to be highly conserved. VviERF6L transcripts were detected in numerous plant organs and were differentially expressed in response to numerous abiotic stresses including water deficit, salinity, and cold as well as biotic stresses such as red blotch virus, N. parvum, and E. necator. VviERF6Ls were differentially expressed across stages of berry development, peaking in the pre-veraison/veraison stage and retaining conserved expression patterns across different vineyards, years, and Vitis cultivars. Co-expression network analysis identified a scarecrow-like transcription factor and a calmodulin-like gene with highly similar expression profiles to the VviERF6L clade. Overexpression of VviERF6L1 in a Seyval Blanc background did not result in detectable morphological phenotypes. Genes differentially expressed in response to VviERF6L1 overexpression were associated with abiotic and biotic stress responses. CONCLUSIONS: VviERF6Ls represent a large and distinct clade of ERF transcription factors in grapevine. The high conservation of protein sequence between these 18 transcription factors may indicate these genes originate from a duplication event in Vitis. Despite high sequence similarity and similar expression patterns, VviERF6Ls demonstrate unique levels of expression supported by similar but heterogeneous promoter sequences. VviERF6L gene expression differed between Vitis species, cultivars and organs including roots, leaves and berries. These genes respond to berry development and abiotic and biotic stresses. VviERF6L1 overexpression in Vitis vinifera results in differential expression of genes related to phytohormone and immune system signaling. Further investigation of this interesting gene family is warranted.

Mediator of tolerance to abiotic stress ERF6 regulates susceptibility of Arabidopsis to Meloidogyne incognita.

Root-knot nematodes transform vascular host cells into permanent feeding structures to selectively withdraw their nutrients from host plants during the course of several weeks. The susceptibility of host plants to root-knot nematode infections is thought to be a complex trait involving many genetic loci. However, genome-wide association (GWA) analysis has so far revealed only four quantitative trait loci (QTLs) linked to the reproductive success of the root-knot nematode Meloidogyne incognita in Arabidopsis thaliana, which suggests that the genetic architecture underlying host susceptibility could be much simpler than previously thought. Here, we report that, by using a relaxed stringency approach in a GWA analysis, we could identify 15 additional loci linked to quantitative variation in the reproductive success of M. incognita in Arabidopsis. To test the robustness of our analysis, we functionally characterized six genes located in a QTL with the lowest acceptable statistical support and smallest effect size. This led us to identify ETHYLENE RESPONSE FACTOR 6 (ERF6) as a novel susceptibility gene for M. incognita in Arabidopsis. ERF6 functions as a transcriptional activator and suppressor of genes in response to various abiotic stresses independent of ethylene signalling. However, whole-transcriptome analysis of nematode-infected roots of the Arabidopsis erf6-1 knockout mutant line showed that allelic variation at this locus may regulate the conversion of aminocyclopropane-1-carboxylate (ACC) into ethylene by altering the expression of 1-aminocyclopropane-1-carboxylate oxidase 3 (ACO3). Our data further suggest that tolerance to abiotic stress mediated by ERF6 forms a novel layer of control in the susceptibility of Arabidopsis to M. incognita.