Repression Motif Research Articles

LkERF6 enhances drought and salt tolerance in transgenic tobacco by regulating ROS homeostasis

The transcription factor Ethylene Responsive Factor (ERF) is crucial for responding to various environmental stressors. Proteins containing the ERF-associated amphiphilic repression (EAR) motif often inhibit gene expression. However, the functions of LkERF, an EAR motif-containing protein from Larix kaempferi, especially in reactive oxygen species (ROS) homeostasis, are not well understood. In the present research, we introduce a novel transcription factor, LkERF6, which contains an EAR motif and positively regulates gene expression, thereby enhancing drought and salt tolerance in tobacco. LkERF6 is classified within the ERF-B1 subfamily due to its conserved AP2/ERF domain and EAR motif. Subcellular localization assays demonstrated LkERF6 is primarily localized in the nucleus. Further analysis revealed that LkERF6 interacts with GCC and DRE elements and is significantly induced by NaCl and PEG6000. Moreover, LkERF6 transgenic tobacco plants exhibit lower ROS accumulation and higher levels of antioxidant enzyme activities. Additionally, correlation analysis identified a strong association between LkERF6 and three genes: LkSOD, LkCCS, and LkCAT. Y1H, EMAS, and DLR assays confirmed that LkERF6 directly interacts with the promoters of these genes through GCC-box and DRE-box to activate their expression. These findings shed new light on the function of EAR motif-containing transcription factors and highlight LkERF6’s crucial role in enhancing abiotic stress resistance by activating multiple ROS clearance genes.

Design and Characterization of a Transcriptional Repression Toolkit for Plants.

Regulation of gene expression is essential for all life. Tools to manipulate the gene expression level have therefore proven to be very valuable in efforts to engineer biological systems. However, there are few well-characterized genetic parts that reduce gene expression in plants, commonly known as transcriptional repressors. We characterized the repression activity of a library consisting of repression motifs from approximately 25% of the members of the largest known family of repressors. Combining sequence information with our trans-regulatory function data, we next generated a library of synthetic transcriptional repression motifs with function predicted in advance. After characterizing our synthetic library, we demonstrated not only that many of our synthetic constructs were functional as repressors but also that our advance predictions of repression strength were better than random guesses. Finally, we assessed the functionality of known transcriptional repression motifs from a wide range of eukaryotes. Our study represents the largest plant repressor motif library experimentally characterized to date, providing unique opportunities for tuning transcription in plants.

AT-hook motif nuclear localized transcription factors function redundantly in promoting root growth through modulation of redox homeostasis.

Maintaining an optimal redox status is essential for plant growth and development, particularly when the plants are under stress. AT-hook motif nuclear localized (AHL) proteins are evolutionarily conserved transcription factors in plants. Much of our understanding about this gene family has been derived from studies on clade A members. To elucidate the functions of clade B genes, we first analyzed their spatial expression patterns using transgenic plants expressing a nuclear localized GFP under the control of their promoter sequences. AHL1, 2, 6, 7, and 10 were further functionally characterized owing to their high expression in the root apical meristem. Through mutant analyses and transgenic studies, we showed that these genes have the ability to promote root growth. Using yeast one-hybrid and dual luciferase assays, we demonstrated that AHL1, 2, 6, 7, and 10 are transcription regulators and this activity is required for their roles in root growth. Although mutants for these genes did not showed obvious defects in root growth, transgenic plants expressing their fusion proteins with the SRDX repressor motif exhibited a short-root phenotype. Through transcriptome analysis, histochemical staining and molecular genetics experiments, we found that AHL10 maintains redox homeostasis via direct regulation of glutathione transferase (GST) genes. When the transcript level of GSTF2, a top-ranked target of AHL10, was reduced by RNAi, the short-root phenotype in the AHL10-SRDX expressing plant was largely rescued. These results together suggest that AHL genes function redundantly in promoting root growth through direct regulation of redox homeostasis.

A transcriptional repressor HVA regulates vascular bundle formation through auxin transport in Arabidopsis stem.

Vascular bundles transport water and photosynthate to all organs, and increased bundle number contributes to crop lodging resistance. However, the regulation of vascular bundle formation is poorly understood in the Arabidopsis stem. We report a novel semi-dominant mutant with high vascular activity, hva-d, showing increased vascular bundle number and enhanced cambium proliferation in the stem. The activation of a C2H2 zinc finger transcription factor, AT5G27880/HVA, is responsible for the hva-d phenotype. Genetic, biochemical, and fluorescent microscopic analyses were used to dissect the functions of HVA. HVA functions as a repressor and interacts with TOPLESS via the conserved Ethylene-responsive element binding factor-associated Amphiphilic Repression motif. In contrast to the HVA activation line, knockout of HVA function with a CRISPR-Cas9 approach or expression of HVA fused with an activation domain VP16 (HVA-VP16) resulted in fewer vascular bundles. Further, HVA directly regulates the expression of the auxin transport efflux facilitator PIN1, as a result affecting auxin accumulation. Genetics analysis demonstrated that PIN1 is epistatic to HVA in controlling bundle number. This research identifies HVA as a positive regulator of vascular initiation through negatively modulating auxin transport and sheds new light on the mechanism of bundle formation in the stem.

The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

Non-transcriptional regulatory activity of SMAX1 and SMXL2 mediates karrikin-regulated seedling response to red light in Arabidopsis

Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways. The transcriptional repressor proteins SUPPRESSOR OF MAX2 1 (SMAX1), SMAX1-like2 (SMXL2), and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or by inhibiting the activities of transcription factors. In this study, we characterized the non-transcriptional regulatory activities of SMXL proteins in Arabidopsis. We discovered that SMAX1 and SMXL2 with mutations in their ethylene-response factor-associated amphiphilic repression (EAR) motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 mutant. SMAX1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR 4 (PIF4) and PIF5 to enhance their protein stability by interacting with phytochrome B (phyB) and suppressing the association of phyB with PIF4 and PIF5. The karrikin-responsive genes were then identified by treatment with GR24ent−5DS, a GR24 analog showing karrikin activity. Interestingly, INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) expression was repressed by GR24ent−5DS treatment in a PIF4- and PIF5-dependent and EAR-independent manner, whereas KARRIKIN UPREGULATED F-BOX 1 (KUF1) expression was induced in a PIF4- and PIF5-independent and EAR-dependent manner. Furthermore, the non-transcriptional regulatory activity of SMAX1, which is independent of the EAR motif, had a global effect on gene expression. Taken together, these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.

The MdMYB44-MdTPR1 repressive complex inhibits MdCCD4 and MdCYP97A3 expression through histone deacetylation to regulate carotenoid biosynthesis in apple.

Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)-mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene-responsive element-binding factor-associated amphiphilic repression (EAR)-containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44-MdTPR1-mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44-mediated carotenoid biosynthesis. MdMYB44 enhanced β-branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co-repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1-MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation-mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR-harboring TF/TPL repressive complex plays a universal role in histone deacetylation-mediated inhibition of gene expression in various plants.

Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber

Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.

SMXL5 attenuates strigolactone signaling in Arabidopsis thaliana by inhibiting SMXL7 degradation

Hormone-activated proteolysis is a recurring theme of plant hormone signaling mechanisms. In strigolactone signaling, the enzyme receptor DWARF14 (D14) and an F-box protein, MORE AXILLARY GROWTH2 (MAX2), mark SUPPRESSOR OF MAX2 1-LIKE (SMXL) family proteins SMXL6, SMXL7, and SMXL8 for rapid degradation. Removal of these transcriptional corepressors initiates downstream growth responses. The homologous proteins SMXL3, SMXL4, and SMXL5, however, are resistant to MAX2-mediated degradation. We discovered that the smxl4 smxl5 mutant has enhanced responses to strigolactone. SMXL5 attenuates strigolactone signaling by interfering with AtD14–SMXL7 interactions. SMXL5 interacts with AtD14 and SMXL7, providing two possible ways to inhibit SMXL7 degradation. SMXL5 function is partially dependent on an ethylene-responsive-element binding-factor-associated amphiphilic repression (EAR) motif, which typically mediates interactions with the TOPLESS family of transcriptional corepressors. However, we found that loss of the EAR motif reduces SMXL5–SMXL7 interactions and the attenuation of strigolactone signaling by SMXL5. We hypothesize that integration of SMXL5 into heteromeric SMXL complexes reduces the susceptibility of SMXL6/7/8 proteins to strigolactone-activated degradation and that the EAR motif promotes the formation or stability of these complexes. This mechanism may provide a way to spatially or temporally fine-tune strigolactone signaling through the regulation of SMXL5 expression or translation.

Lignin biosynthesis pathway repressors in gymnosperms: differential repressor domains as compared to angiosperms.

Lignin is a polyphenolic polymer present in the cell walls of specialized plant cell types in vascular plants that provides structural support and plays a major role in plant protection. The lignin biosynthesis pathway is regulated by transcription factors from the MYB (myeloblastosis) family. While several MYB members positively regulate lignin synthesis, only a few negatively regulate lignin synthesis. These lignin suppressors are well characterized in model plant species; however, their role has not been fully explored in gymnosperms. Lignin forms one of the major hurdles for the forest-based industry e.g. paper, pulp, and biofuel production. Therefore, the detailed mechanisms involved in the regulation of lignin synthesis are valuable, especially in conifers that form the major source of softwood for timber and paper production. In this review, the potential and differential domains present in the MYB suppressors in gymnosperms are discussed, along with their phylogenetic analysis. Sequence analysis revealed that the N-terminal regions of the MYB suppressor members were found to be conserved among the gymnosperms and angiosperms containing the R2, R3, and bHLH domains, while the C-terminal regions were found to be highly variable. The typical repressor motifs like the LxLxL-type EAR motif and the TLLLFR motif were absent from the C-terminal regions of MYB suppressors from most gymnosperms. However, although the gymnosperms lacked the characteristic repressor domains, a R2R3-type MYB member from Ginkgo was reported to repress the lignin biosynthetic pathway. It is proposed that gymnosperms possess unique kinds of repressors that need further functional validation.

Genome editing and molecular analyses of an Arabidopsis transcription factor, LATE FLOWERING.

Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.

Cell-type-directed design of synthetic enhancers.

Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes1. It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. Here we show that deep learning models2-6, can be used to efficiently design synthetic, cell-type-specific enhancers, starting from random sequences, and that this optimization process allows detailed tracing of enhancer features at single-nucleotide resolution. We evaluate the function of fully synthetic enhancers to specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. We further exploit enhancer design to create 'dual-code' enhancers that target two cell types and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the state space searches towards local optima, we characterize enhancer codes through the strength, combination and arrangement of transcription factor activator and transcription factor repressor motifs. Finally, we apply the same strategies to successfully design human enhancers, which adhere to enhancer rules similar to those of Drosophila enhancers. Enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states.

The fungal pathogen Ustilago maydis targets the maize corepressor RELK2 to modulate host transcription for tumorigenesis.

Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection. In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N-terminus of RELK2 through its two Ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of RELK2. We propose that Tip6 mimics the recruitment of RELK2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6. Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor RELK2 to disrupt the transcriptional networks of the host plant.

Repression motif in HSF1 regulated by phosphorylation

The heat shock factor 1 (HSF1) is a transcription factor that itself is a sensor for stress and integrates various intrinsic or environmental stress sensing pathways. Thus HSF1 orchestrates the heat shock response (HSR) by translating these pathways into a distinct transcriptional program that aids the cells to cope with and adapt to proteotoxic stress. Although heavily researched the regulation of HSF1 activation is still not completely understood. A conserved reaction to stress is the hyperphosphorylation of the otherwise confined constitutive phosphorylated HSF1. Therefore, this stress specific phosphorylation is believed to be involved in the regulatory mechanism and hence, was and is focus of many studies, ascribing various effects to single phosphorylation sites. To gain additional insight into effects of phosphorylation, HSF1 carrying amino acid substitutions on up to 18 amino acids were tested for their transactivation potential on an HSR reporter plasmid. A pattern of eleven phosphor-mimicking and diminishing amino acid substitutions on well-known phosphorylation sites of HSF1 were introduced to produce transcriptional active [11 M(+)] or repressed [11 M(−)] phenotypes. It could be confirmed that heat activates HSF1 regardless of phosphorylation. Distinct cellular stress, obtained by chemical HSR inducers or mimicked by a constitutively active HSF1, showed clear differences in the activation potential of HSF1-11 M(+) and 11 M(−). Further refinement to the single amino acid level identified the S303/307 double-phosphorylation motif, wherein phosphorylation of S303 was sole responsible for the repressing effect. The effect could be reproduced in different cell lines and is not entirely based on degradation. A small repression motif could be dissociated from the HSF1 context, which is still capable of repressing the background transcription of a specifically designed reporter plasmid. Taken together these results indicate, that besides already described mechanisms of pS303/307 mediated repression of HSF1 activation, an additional mechanism repressing the transcriptional output of the entire HSE containing promoter is mediated by this small repressive motif.

Multi-omics atlas of combinatorial abiotic stress responses in wheat.

Field-grown crops rarely experience growth conditions in which yield can be maximized. Environmental stresses occur in combination, with advancements in crop tolerance further complicated by its polygenic nature. Strategic targeting of causal genes is required to meet future crop production needs. Here, we employed a systems biology approach in wheat (Triticum aestivum L.) to investigate physio-metabolic adjustments and transcriptome reprogramming involved in acclimations to heat, drought, salinity and all combinations therein. A significant shift in magnitude and complexity of plant response was evident across stress scenarios based on the agronomic losses, increased proline concentrations and 8.7-fold increase in unique differentially expressed transcripts (DETs) observed under the triple stress condition. Transcriptome data from all stress treatments were assembled into an online, open access eFP browser for visualizing gene expression during abiotic stress. Weighted gene co-expression network analysis revealed 152 hub genes of which 32% contained the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) transcriptional repression motif. Cross-referencing against the 31 DETs common to all stress treatments isolated TaWRKY33 as a leading candidate for greater plant tolerance to combinatorial stresses. Integration of our findings with available literature on gene functional characterization allowed us to further suggest flexible gene combinations for future adaptive gene stacking in wheat. Our approach demonstrates the strength of robust multi-omics-based data resources for gene discovery in complex environmental conditions. Accessibility of such datasets will promote cross-validation of candidate genes across studies and aid in accelerating causal gene validation for crop resiliency.

CtBP: A global regulator of balancing acts and homeostases.

The classical role of C-terminal binding protein (CtBP) is that of a global corepressor. However, its exact mechanism of repression is not known. In this review, we elucidate the repression motif used by CtBP. Further, we provide other unifying features of its mechanism of action. For example, in the presence of a high NADH/NAD+ ratio in the cell, causing a low glycolytic condition, the NADH-bound dimeric form of CtBP causes global repression, maintaining balances and homeostases of many cellular processes, under the cell surveillance of p53 and NFkB. In contrast, in the presence of a low NADH/NAD+ ratio, causing a high glycolytic condition, the NADH-free monomeric form of CtBP blocks p53 function and NFkB-mediated transcription. Further, a low NADH/NAD+ ratio upsets the homeostases and balances in the absence of the cell surveillances of p53 and NFkB, causing global instability, the dominant outcome of CtBP's action in carcinogenesis, in cells in a high glycolytic state.

Repressive ZINC FINGER OF ARABIDOPSIS THALIANA proteins promote programmed cell death in the Arabidopsis columella root cap.

Developmental programmed cell death (dPCD) controls a plethora of functions in plant growth and reproduction. In the root cap of Arabidopsis (Arabidopsis thaliana), dPCD functions to control organ size in balance with the continuous stem cell activity in the root meristem. Key regulators of root cap dPCD including SOMBRERO/ANAC033 (SMB) belong to the NAC family of transcription factors. Here, we identify the C2H2 zinc finger protein ZINC FINGER OF ARABIDOPSIS THALIANA 14 ZAT14 as part of the gene regulatory network of root cap dPCD acting downstream of SMB. Similar to SMB, ZAT14-inducible misexpression leads to extensive ectopic cell death. Both the canonical EAR motif and a conserved L-box motif of ZAT14 act as transcriptional repression motifs and are required to trigger cell death. While a single zat14 mutant does not show a cell death-related phenotype, a quintuple mutant knocking out 5 related ZAT paralogs shows a delayed onset of dPCD execution in the columella and the adjacent lateral root cap. While ZAT14 is co-expressed with established dPCD-associated genes, it does not activate their expression. Our results suggest that ZAT14 acts as a transcriptional repressor controlling a so far uncharacterized subsection of the dPCD gene regulatory network active in specific root cap tissues.

The transcription factor POPEYE negatively regulates the expression of bHLH Ib genes to maintain iron homeostasis.

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency-responsive genes to promote Fe uptake. POPEYE (PYE) is a key bHLH (basic helix-loop-helix) transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive in Arabidopsis. We found that the overexpression of PYE attenuates the expression of Fe deficiency-responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by associating with their promoters. Although PYE contains an ethylene response factor-associated amphiphilic repression (EAR) motif, it does not interact with the transcriptional co-repressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Sub-cellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a nuclear export signal (NES) which is required for the cytoplasmic localization of PYE. Mutation of the NES amplifies the repression function of PYE, resulting in down-regulation of Fe deficiency-responsive genes. Co-expression assays indicated that three bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses the transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides new insights into the Fe deficiency response signalling pathway and enhances the understanding of PYE functions in Arabidopsis.

ETHYLENE RESPONSE FACTORS 4.1/4.2 with an EAR motif repress anthocyanin biosynthesis in red-skinned pears.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of 'Starkrimson' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

EcoGothic Gardens in the Long Nineteenth Century: Phantoms, Fantasy and Uncanny Flowers, edited by Sue Edney

Reviewed by: EcoGothic Gardens in the Long Nineteenth Century: Phantoms, Fantasy and Uncanny Flowers ed. by Sue Edney Lindsay Wells (bio) EcoGothic Gardens in the Long Nineteenth Century: Phantoms, Fantasy and Uncanny Flowers, edited by Sue Edney; pp. xviii + 217. Manchester: Manchester University Press, 2020, £85.00. EcoGothic Gardens in the Long Nineteenth Century: Phantoms, Fantasy and Uncanny Flowers begins its analysis of horticulture, literature, and environmental thought by questioning the apparent incompatibility of its objects of study: the orderly garden and the chaotic Gothic. As editor Sue Edney observes in her introduction to the volume, the “human dominance” that seemingly defines horticultural space starts to dissipate when viewed through the lens of the Gothic—an expressive mode that “allows us to question human ability to control events, people or even places.” The essays that comprise this collection explore this process from a diverse range of perspectives, from landscape studies and history of science to new materialism and the plant humanities. Each chapter brings fresh attention to the ecological and Gothic undercurrents of nineteenth-century gardens, resulting in a compelling exposition of not only the term ecoGothic, but also the “uncanny deeds and presences” that haunt the archives of horticultural history (1). As its title suggests, this volume connects recent ecocritical readings of Gothic narrative to both real and imaginary gardens of the nineteenth century. While the book features essays on German and American literature, the majority of its chapters concentrate on British texts, with a particular emphasis on Victorian fiction. The project builds on Andrew Smith and William Hughes’s edited collection Ecogothic (2013), as well as the 2014 special issue of Gothic Studies (1999–present) on the same subject. The contributors to Edney’s volume take these earlier studies in new directions by identifying the garden as a vital component of the ecoGothic paradigm, which interprets monstrosity, repression, and other Gothic motifs from an environmental angle. The prominence of uncanny plants and landscapes in British culture comes into clear focus throughout this collection, with each author demonstrating the ways in which nineteenth-century gardens afford thought-provoking explorations of the natural world. One of the key contributions that EcoGothic Gardens makes to Victorian studies is the link it forges between new materialism and critical plant studies—two schools of thought with a shared interest in the nonhuman. The impact of new materialism and material ecocriticism on recent analyses of Victorian culture has been significant, and many of the contributors to EcoGothic Gardens engage with this discourse. Shelley Saguaro, for instance, reads the gardens in Nathaniel Hawthorne’s “Rappaccini’s Daughter” (1844) and The Blithedale Romance (1852) alongside Donna Haraway’s concept of the Chthulucene. In a similar respect, Teresa Fitzpatrick connects Stacy Alaimo’s work on trans-corporeality to carnivorous plant fiction from the fin de siècle. Other chapters, meanwhile, draw on insights from Jane Bennett, Karen Barad, Serenella Iovino, and Serpil Oppermann. EcoGothic Gardens sustains the ongoing exchange between new materialism and Victorian studies, yet it also embraces related scholarship on the animacy of plants. Several of the book’s contributors engage with Dawn Keetley and Angela Tenga’s work on plant horror, Monica Gagliano’s research on vegetal communication, and Michael Marder’s theories on plants and philosophy. EcoGothic Gardens demonstrates the ways in which reinterpretations of material and vegetal vibrancy might be brought into productive dialogue with one another, especially when dealing with Victorian horticulture. [End Page 720] Even the most regimented of gardens examined in this book threaten to undermine the will of their human caretakers with a vegetal vitality that exceeds all control. In her essay on the early-nineteenth-century fiction of Joseph von Eichendorff and Johann Wolfgang von Goethe, Heather I. Sullivan refines the concept of the ecoGothic into what she terms “the ‘Gothic green,’” or instances when “the human is, in some way, subsumed by vegetal forces” (17). The collaboration (and confrontation) between human and vegetal agents in gardens continually resurfaces throughout this book, including Edney’s chapter on Alfred, Lord Tennyson’s “Mariana” (1830) and Maud (1855). “In gardens,” Edney writes, “nature and culture are conspirators as well as co-workers” (168). This observation lies at the conceptual...