Arabidopsis Transcription Factor Research Articles

Expressing banana transcription factor MaERFVII3 in Arabidopsis confers enhanced waterlogging tolerance and root growth.

Waterlogging poses a significant threat to plant growth and yield worldwide. Identifying the genes responsible for mitigating waterlogging stress is crucial. Ethylene-responsive factors (ERFs) are transcriptional regulators that respond to various biotic and abiotic stresses in plants. However, their roles and involvement in responding to waterlogging stress remain largely unexplored. Hence, this study aimed to elucidate the role of ERFs in enhancing banana plant resilience to waterlogging. We hypothesized that introducing a group VII ERF transcription factor in Arabidopsis could enhance waterlogging stress tolerance. To test this hypothesis, we isolated MaERFVII3 from banana roots, where it exhibited a significant induction in response to waterlogging stress. The isolated MaERFVII3 was introduced into Arabidopsis plants for functional gene studies. Compared with wild-type plants, the MaERFVII3-expressing Arabidopsis showed increased survival and biomass under waterlogging stress. Furthermore, the abundance of transcripts related to waterlogging and hypoxia response showed an elevation in transgenic plants but a decrease in wild-type and empty vector plants when exposed to waterlogging stress. Our results demonstrate the significant contribution of MaERFVII3 to waterlogging tolerance in Arabidopsis, providing baseline data for further exploration and potentially contributing to crop improvement programs.

A nuclear WD40 repeat protein PRL1 regulates stability of MYB4 transcription factor in Arabidopsis

MYB4, a member of the R2R3-type subfamily of MYB transcription factor plays a crucial role in regulating the accumulation of UV-B absorbing phenylpropanoids in plants. UV-B exposure for a longer duration down-regulates the expression of MYB4 gene in Arabidopsis. MYB4 protein represses its own expression by binding to its own promoter. However, at present practically nothing is known about the post-translational regulation of MYB4 protein in vivo. Here, we provide evidence that in Arabidopsis MYB4 protein is phosphorylated in vivo and is targeted by the ubiquitin-26S proteasome-dependent pathway. Immunoprecipitation, immunoblotting, and phosphoprotein staining experiments have revealed that both the accumulation pattern and phosphorylation of MYB4 increase in the light condition during the 24 hours time span under long-day conditions. Yeast two-hybrid and bimolecular fluorescence complementation assays have shown that MYB4 directly interacts with a nuclear WD40 repeat protein, PRL1 in vivo. Cell-free protein degradation assay in the absence and presence of proteasome inhibitor indicates that MYB4 is degraded in a ubiquitin proteasome-dependent manner. Furthermore, analyses of MYB4 protein accumulation levels in transgenic atmyb4–1 mutant line expressing 35 S:AtMYB4 (35 S:AtMYB4-atmyb4–1) and atprl1–1 mutant line indicate that PRL1 regulate stability of MYB4 in Arabidopsis. Overall, our results provide important information on the possible mechanism of post-translational modification and regulation of stability of MYB4 protein in Arabidopsis in vivo.

Complex control of seed germination timing by ERF50 involves RGL2 antagonism and negative feedback regulation of DOG1.

Seed dormancy governs germination timing, with both evolutionary and applied consequences. Despite extensive studies on the hormonal and genetic control of these processes, molecular mechanisms directly linking dormancy and germination remain poorly understood. By screening a collection of lines overexpressing Arabidopsis transcription factors, we identified ERF50 as a key gene to control dormancy and germination. To study its regulation, we measured seed-related physiological parameters in loss-of-function mutants and carried out transactivation, protein interaction and ChIP-PCR analyses. We found direct ERF50-mediated repression of DOG1 and activation of EXPA2 transcription, which results in enhanced seed germination. Although ERF50 expression is increased by DOG1 in dormant seeds, ERF50 germination-promoting activity is blocked by RGL2. The physiological, genetic and molecular evidence gathered here supports that ERF50 controls germination timing by regulating DOG1 levels to leverage its role as enhancer of seed germination, via RGL2 antagonism on EXPA2 expression. Our results highlight the central role of ERF50 as a feedback regulator to couple and fine-tune seed dormancy and germination.

Arabidopsis transcription factor STOP1 directly activates expression of NOD26-LIKE MAJOR INTRINSIC PROTEIN5;1, and is involved in the regulation of tolerance to low-boron stress.

Transcriptional regulation is a crucial component of plant adaptation to numerous different stresses; however, its role in how plants adapt to low-boron (B) stress remains unclear. In this study, we show that the C2H2-type transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) in Arabidopsis is essential for improving plant growth under low-B conditions. STOP1 and the boric acid-channel protein NOD26-LIKE MAJOR INTRINSIC PROTEIN5;1 (NIP5;1) were found to co-localize in root epidermal cells, and STOP1 binds to the 5´-untranslated region of NIP5;1 to activate its expression and enhance B uptake by the roots. Overexpression of STOP1 increased tolerance to low-B stress by up-regulating NIP5;1 transcript levels. Further genetic analyses revealed that STOP1 and NIP5;1 function together in the same pathway to confer low-B tolerance. These results highlight the importance of the STOP1-NIP5;1 module in improving plant growth under low-B conditions.

LsMybW-encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed

Key messageWe identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars.Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black—in its wild ancestor—to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between “ShinanoPower” (white) and “Escort” (black). The white seed trait was controlled by a single recessive locus (48.055–50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of “ShinanoPower” and “Escort.” Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativaMyb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.

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.

Environmental regulation of male fertility is mediated through Arabidopsis transcription factors bHLH89, 91, and 10.

Formation of functional pollen and successful fertilization rely on the spatial and temporal regulation of anther and pollen development. This process responds to environmental cues to maintain optimal fertility despite climatic changes. Arabidopsis transcription factors basic helix-loop-helix (bHLH) 10, 89, and 91 were previously thought to be functionally redundant in their control of male reproductive development, however here we show that they play distinct roles in the integration of light signals to maintain pollen development under different environmental conditions. Combinations of the double and triple bHLH10,89,91 mutants were analysed under normal (200 μmol m-2 s-1) and low (50 μmol m-2 s-1) light conditions to determine the impact on fertility. Transcriptomic analysis of a new conditionally sterile bhlh89,91 double mutant shows differential regulation of genes related to sexual reproduction, hormone signal transduction, and lipid storage and metabolism under low light. Here we have shown that bHLH89 and bHLH91 play a role in regulating fertility in response to light, suggesting that they function in mitigating environmental variation to ensure fertility is maintained under environmental stress.

An miR164-resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development.

The sex determination process in cucurbits involves the control of stamen or carpel development during the specification of male or female flowers from a bisexual floral meristem, a function coordinated by ethylene. A gain-of-function mutation in the miR164-binding site of CpCUC2B, ortholog of the Arabidopsis transcription factor gene CUC2, not only produced ectopic floral meristems and organs, but also suppressed the development of carpels and promoted the development of stamens. The cuc2b mutation induced the transcription of CpCUC2B in the apical shoots of plants after female flowering but repressed other CUC genes regulated by miR164, suggesting a conserved functional redundancy of these genes in the development of squash flowers. The synergistic androecious phenotype of the double mutant between cuc2b and etr2b, an ethylene-insensitive mutation that enhances the production of male flowers, demonstrated that CpCUC2B arrests the development of carpels independently of ethylene and CpWIP1B. The transcriptional regulation of CpCUC1, CpCUC2, and ethylene genes in cuc2b and ethylene mutants also confirms this conclusion. However, the epistasis of cuc2b over aco1a, a mutation that suppresses stamen arrest in female flowers, and the down-regulation of CpACS27A in cuc2b female apical shoots, indicated that CpCUC2B promotes stamen development by suppressing the late ethylene production.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Large-scale identification of novel transcriptional regulators of the aliphatic glucosinolate pathway in Arabidopsis.

Aliphatic glucosinolates are a large group of plant secondary metabolites characteristic of Brassicaceae, including the model plant Arabidopsis. The diverse and complex degradation products of aliphatic glucosinolates contribute to plant responses to herbivory, pathogen attack, and environmental stresses. Most of the biosynthesis genes in the aliphatic glucosinolate pathway have been cloned in Arabidopsis, and the research focus has recently shifted to the regulatory mechanisms controlling aliphatic glucosinolate accumulation. Up till now, more than 40 transcriptional regulators have been identified as regulating the aliphatic glucosinolate pathway, but many more novel regulators likely remain to be discovered based on research evidence over the past decade. In the current study, we took a systemic approach to functionally test 155 candidate transcription factors in Arabidopsis identified by yeast one-hybrid assay, and successfully validated at least 30 novel regulators that could significantly influence the accumulation of aliphatic glucosinolates in our experimental set-up. We also showed that the regulators of the aliphatic glucosinolate pathway have balanced positive and negative effects, and glucosinolate metabolism and plant development can be coordinated. Our work is the largest scale effort so far to validate transcriptional regulators of a plant secondary metabolism pathway, and provides new insights into how the highly diverse plant secondary metabolism is regulated at the transcriptional level.

Arabidopsis transcription factor WRKY45 confers cadmium tolerance via activating PCS1 and PCS2 expression

Cadmium (Cd) has long been recognized as toxic pollutant to crops worldwide. The biosynthesis of glutathione-dependent phytochelatin (PC) plays crucial roles in the detoxification of Cd in plants. However, its regulatory mechanism remains elusive. Here, we revealed that Arabidopsis transcription factor WRKY45 confers Cd tolerance via promoting the expression of PC synthesis-related genes PCS1 and PCS2, respectively. Firstly, we found that Cd stress induces the transcript levels of WRKY45 and its protein abundance. Accordingly, in contrast to wild type Col-0, the increased sensitivity to Cd is observed in wrky45 mutant, while overexpressing WRKY45 plants are more tolerant to Cd. Secondly, quantitative real-time PCR revealed that the expression of AtPCS1 and AtPCS2 is stimulated in overexpressing WRKY45 plants, but decreased in wrky45 mutant. Thirdly, WRKY45 promotes the expression of PCS1 and PCS2, electrophoresis mobility shift assay analysis uncovered that WRKY45 directly binds to the W-box cis-element of PCS2 promoter. Lastly, the overexpression of WRKY45 in Col-0 leads to more accumulation of PCs in Arabidopsis, and the overexpression of PCS1 or PCS2 in wrky45 mutant plants rescues the phenotypes induced by Cd stress. In conclusion, our results show that AtWRKY45 positively regulates Cd tolerance in Arabidopsis via activating PCS1 and PCS2 expression.

Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence.

Senescence is a highly regulated process driven by developmental age and environmental factors. Although leaf senescence is accelerated by nitrogen (N) deficiency, the underlying physiological and molecular mechanisms are largely unknown. Here, we reveal that BBX14, a previously uncharacterized BBX-type transcription factor in Arabidopsis, is crucial for N starvation-induced leaf senescence. We find that inhibiting BBX14 by artificial miRNA (amiRNA) accelerates senescence during N starvation and in darkness, while BBX14 overexpression (BBX14-OX) delays it, identifying BBX14 as a negative regulator of N starvation- and dark-induced senescence. During N starvation, nitrate and amino acids like glutamic acid, glutamine, aspartic acid, and asparagine were highly retained in BBX14-OX leaves compared to the wild type. Transcriptome analysis showed a large number of senescence-associated genes (SAGs) to be differentially expressed between BBX14-OX and wild-type plants, including ETHYLENE INSENSITIVE3 (EIN3) which regulates N signaling and leaf senescence. Chromatin immunoprecipitation (ChIP) showed that BBX14 directly regulates EIN3 transcription. Furthermore, we revealed the upstream transcriptional cascade of BBX14. By yeast one-hybrid screen and ChIP, we found that MYB44, a stress-responsive MYB transcription factor, directly binds to the promoter of BBX14 and activates its expression. In addition, Phytochrome Interacting Factor 4 (PIF4) binds to the promoter of BBX14 to repress BBX14 transcription. Thus, BBX14 functions as a negative regulator of N starvation-induced senescence through EIN3 and is directly regulated by PIF4 and MYB44.

The nitrate-responsive transcription factor MdNLP7 regulates callus formation by modulating auxin response

Under appropriate culture conditions, plant cells can regenerate new organs or even whole plants. De novo organ regeneration is an excellent biological system, which usually requires additional growth regulators, including auxin and cytokinin. Nitrate is an essential nutrient element for plant vegetative and reproductive development. It has been reported that nitrate is involved in auxin biosynthesis and transport throughout the growth and development of plants. In this study, we demonstrated that the ectopic expression of the MdNLP7 transcription factor in Arabidopsis could regulate the regeneration of root explants. MdNLP7 mainly participated in the regulation of callus formation, starting with pericycle cell division, and mainly affected auxin distribution and accumulation in the regulation process. Moreover, MdNLP7 upregulated the expression of genes related to auxin biosynthesis and transport in the callus formation stage. The results demonstrated that MdNLP7 may play a role in the nitrate-modulated regeneration of root explants. Moreover, the results revealed that nitrate–auxin crosstalk is required for de novo callus initiation and clarified the mechanisms of organogenesis.

Overexpression of TaMYB4 Confers Freezing Tolerance in Arabidopsis thaliana.

Freezing stress is one of the main factors limiting the growth and yield of wheat. In this study, we found that TaMYB4 expression was significantly upregulated in the tillering nodes of the strong cold-resistant winter wheat variety Dongnongdongmai1 (Dn1) under freezing stress. Weighted gene co-expression network analysis, qRT-PCR and protein-DNA interaction experiments demonstrated that monodehydroascorbate reductase (TaMDHAR) is a direct target of TaMYB4. The results showed that overexpression of TaMYB4 enhanced the freezing tolerance of transgenic Arabidopsis. In TaMYB4 overexpression lines (OE-TaMYB4), AtMDHAR2 expression was upregulated and ascorbate-glutathione (AsA-GSH) cycle operation was enhanced. In addition, the expression of cold stress marker genes such as AtCBF1, AtCBF2, AtCBF3, AtCOR15A, AtCOR47, AtKIN1 and AtRD29A in OE-TaMYB4 lines was significantly upregulated. Therefore, TaMYB4 may increase freezing tolerance as a transcription factor (TF) in Arabidopsis through the AsA-GSH cycle and DREB/CBF signaling pathway. This study provides a potential gene for molecular breeding against freezing stress.

Cold-responsive transcription factors in Arabidopsis and rice: A regulatory network analysis using array data and gene co-expression network.

Plant growth and development can be influenced by cold stress. Responses of plants to cold are regulated in part by transcription factors (TFs) and microRNAs, which their determination would be necessary in comprehension of the corresponding molecular cues. Here, transcriptomes of Arabidopsis and rice were analyzed to computationally determine TFs and microRNAs that are differentially responsive to cold treatment, and their co-expression networks were established. Among 181 Arabidopsis and 168 rice differentially expressed TF genes, 37 (26 novel) were up- and 16 (8 novel) were downregulated. Common TF encoding genes were from ERF, MYB, bHLH, NFY, bZIP, GATA, HSF and WRKY families. NFY A4/C2/A10 were the significant hub TFs in both plants. Phytohormone responsive cis-elements such as ABRE, TGA, TCA and LTR were the common cis-elements in TF promoters. Arabidopsis had more responsive TFs compared to rice possibly due to its greater adaptation to ranges geographical latitudes. Rice had more relevant miRNAs probably because of its bigger genome size. The interacting partners and co-expressed genes were different for the common TFs so that of the downstream regulatory networks and the corresponding metabolic pathways. Identified cold-responsive TFs in (A + R) seemed to be more engaged in energy metabolism esp. photosynthesis, and signal transduction, respectively. At post-transcriptional level, miR5075 showed to target many identified TFs in rice. In comparison, the predictions showed that identified TFs are being targeted by diverse groups of miRNAs in Arabidopsis. Novel TFs, miRNAs and co-expressed genes were introduced as cold-responsive markers that can be harnessed in future studies and development of crop tolerant varieties.

A complex tissue-specific interplay between the Arabidopsis transcription factors AtMYB68, AtHB23, and AtPHL1 modulates primary and lateral root development and adaptation to salinity.

Adaptation to different soil conditions is a well-regulated process vital for plant life. AtHB23 is a homeodomain-leucine zipper I transcription factor (TF) that was previously revealed as crucial for plant survival under salinity conditions. We wondered whether this TF has partners to perform this essential function. Therefore, TF cDNA library screening, yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays were complemented with expression analyses and phenotypic characterization of silenced, mutant, overexpression, and crossed plants in normal and salinity conditions. We revealed that AtHB23, AtPHL1, and AtMYB68 interact with each other, modulating root development and the salinity response. The encoding genes are coexpressed in specific root tissues and at specific developmental stages. In normal conditions, amiR68 silenced plants have fewer initiated roots, the opposite phenotype to that shown by amiR23 plants. AtMYB68 and AtPHL1 play opposite roles in lateral root elongation. Under salinity conditions, AtHB23 plays a crucial positive role in cooperating with AtMYB68, whereas AtPHL1 acts oppositely by obstructing the function of the former, impacting the plant's survival ability. Such interplay supports the complex interaction between these TF in primary and lateral roots. The root adaptation capability is associated with the amyloplast state. We identified new molecular players that through a complex relationship determine Arabidopsis root architecture and survival in salinity conditions.

MbNAC22, a Malus baccata NAC Transcription Factor, Increased Drought and Salt Tolerance in Arabidopsis

As an excellent grafting material, Malus baccata (L.) Borkh is native to Liaoning, Jilin, Heilongjiang and other regions in China, with a strong adverse environmental adaptability. As a typical transcription factor, the NAC gene acts as a regulator in many molecular pathways responding to abiotic stress. However, research of NAC in the Malus baccata has just begun. In the present research, a new NAC transcription factor, MbNAC22, was obtained from the seedlings of Malus baccata, and its function in drought and salt treatments was studied by heterologous expression. The open reading frame of the MbNAC22 gene is 768, encoding 255 amino acids (aa). Through confocal microscopy, MbNAC22 was found to be located in the nucleus. The heterologous expression of MbNAC22 in Arabidopsis showed that it enhanced the viability of Arabidopsis under drought and salt treatments. Under stresses, the chlorophyll content of the plants decreased, but the decline of the overexpressed-MbNAC22 Arabidopsis was relatively low. Through phenotypic observation and determination of stress-related physiological indicators, it was found that compared with WT Arabidopsis, overexpressed-MbNAC22 Arabidopsis had a higher tolerance to stresses. Under stresses, the overexpression of MbNAC22 positively regulated ion-transport-related genes (AtNHX1 and AtSOS1), the key genes of the ABA pathway (AtNCED3 and AtDREB2A), the proline synthesis gene (AtP5CS2) and the drought-induced gene (AtERD11), while the expression of the leaf senescence-associated gene (AtSAG21) and programmed cell death related gene (AtAEP1) was inhibited. Therefore, we speculate that MbNAC22 responds positively to drought and salt stresses by regulating the expression of stress-related genes.

TSPTFBS 2.0: trans-species prediction of transcription factor binding sites and identification of their core motifs in plants.

An emerging approach using promoter tiling deletion via genome editing is beginning to become popular in plants. Identifying the precise positions of core motifs within plant gene promoter is of great demand but they are still largely unknown. We previously developed TSPTFBS of 265 Arabidopsis transcription factor binding sites (TFBSs) prediction models, which now cannot meet the above demand of identifying the core motif. Here, we additionally introduced 104 maize and 20 rice TFBS datasets and utilized DenseNet for model construction on a large-scale dataset of a total of 389 plant TFs. More importantly, we combined three biological interpretability methods including DeepLIFT, in-silico tiling deletion, and in-silico mutagenesis to identify the potential core motifs of any given genomic region. For the results, DenseNet not only has achieved greater predictability than baseline methods such as LS-GKM and MEME for above 389 TFs from Arabidopsis, maize and rice, but also has greater performance on trans-species prediction of a total of 15 TFs from other six plant species. A motif analysis based on TF-MoDISco and global importance analysis (GIA) further provide the biological implication of the core motif identified by three interpretability methods. Finally, we developed a pipeline of TSPTFBS 2.0, which integrates 389 DenseNet-based models of TF binding and the above three interpretability methods. TSPTFBS 2.0 was implemented as a user-friendly web-server (http://www.hzau-hulab.com/TSPTFBS/), which can support important references for editing targets of any given plant promoters and it has great potentials to provide reliable editing target of genetic screen experiments in plants.

Transcription factor bZIP52 modulates Arabidopsis seed oil biosynthesis through interaction with WRINKLED1.

Transcriptional regulation mediated by combinatorial interaction of transcription factors (TFs) is a key molecular mechanism modulating plant development and metabolism. Basic leucine zipper (bZIP) TFs play important roles in various plant developmental and physiological processes. However, their involvement in fatty acid biosynthesis is largely unknown. Arabidopsis (Arabidopsis thaliana) WRINKLED1 (WRI1) is a pivotal TF in regulation of plant oil biosynthesis and interacts with other positive and negative regulators. In this study, we identified two bZIP TFs, bZIP21 and bZIP52, as interacting partners of AtWRI1 by yeast-two-hybrid (Y2H)-based screening of an Arabidopsis TF library. We found that coexpression of bZIP52, but not bZIP21, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves. The AtWRI1-bZIP52 interaction was further verified by Y2H, in vitro pull-down, and bimolecular fluorescence complementation assays. Transgenic Arabidopsis plants overexpressing bZIP52 showed reduced seed oil accumulation, while the CRISPR/Cas9-edited bzip52 knockout mutant exhibited increased seed oil accumulation. Further analysis revealed that bZIP52 represses the transcriptional activity of AtWRI1 on the fatty acid biosynthetic gene promoters. Together, our findings suggest that bZIP52 represses fatty acid biosynthesis genes through interaction with AtWRI1, resulting in a reduction of oil production. Our work reports a previously uncharacterized regulatory mechanism that enables fine-tuning of seed oil biosynthesis.

Impact of Light and Dark Treatment on Phenylpropanoid Pathway Genes, Primary and Secondary Metabolites in Agastache rugosa Transgenic Hairy Root Cultures by Overexpressing Arabidopsis Transcription Factor AtMYB12.

Agastache rugosa, otherwise called Korean mint, has a wide range of medicinal benefits. In addition, it is a rich source of several medicinally valuable compounds such as acacetin, tilianin, and some phenolic compounds. The present study aimed to investigate how the Tartary buckwheat transcription factor AtMYB12 increased the primary and secondary metabolites in Korean mint hairy roots cultured under light and dark conditions. A total of 50 metabolites were detected by using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The result showed that the AtMYB12 transcription factor upregulated the phenylpropanoid biosynthesis pathway genes, which leads to the highest accumulation of primary and secondary metabolites in the AtMYB12-overexpressing hairy root lines (transgenic) than that of the GUS-overexpressing hairy root line (control) when grown under the light and dark conditions. However, when the transgenic hairy root lines were grown under dark conditions, the phenolic and flavone content was not significantly different from that of the control hairy root lines. Similarly, the heat map and hierarchical clustering analysis (HCA) result showed that most of the metabolites were significantly abundant in the transgenic hairy root cultures grown under light conditions. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed that the identified metabolites were separated far based on the primary and secondary metabolite contents present in the control and transgenic hairy root lines grown under light and dark conditions. Metabolic pathway analysis of the detected metabolites showed 54 pathways were identified, among these 30 were found to be affected. From these results, the AtMYB12 transcription factor activity might be light-responsive in the transgenic hairy root cultures, triggering the activation of the primary and secondary metabolic pathways in Korean mint.