Arabidopsis Thaliana Transcription Factor Research Articles

Arabidopsis WRKY1 promotes monocarpic senescence by integrative regulation of flowering, leaf senescence, and nitrogen remobilization

Monocarpic senescence, characterized by whole-plant senescence following a single flowering phase, is widespread in seed plants, particularly in crops, determining seed harvest time and quality. However, how external and internal signals are systemically integrated into monocarpic senescence remains largely unknown. Here, we report that the Arabidopsis thaliana transcription factor WRKY1 plays essential roles in multiple key steps of monocarpic senescence. WRKY1 expression is induced by age, salicylic acid (SA), and nitrogen (N) deficiency. Flowering and leaf senescence are accelerated in the WRKY1 overexpression lines but are delayed in the wrky1 mutants. The combined DNA affinity purification sequencing and RNA sequencing analyses uncover the direct target genes of WRKY1. Further studies show that WRKY1 coordinately regulates three processes in monocarpic senescence: (1) suppressing FLOWERING LOCUS C gene expression to initiate flowering, (2) inducing SA biosynthesis genes to promote leaf senescence, and (3) activating the N assimilation and transport genes to trigger N remobilization. In summary, our study reveals how one stress-responsive transcription factor, WRKY1, integrates flowering, leaf senescence, and N remobilization processes into monocarpic senescence, providing important insights into plant lifetime regulation.

Genetic control of thermomorphogenesis in tomato inflorescences

Understanding how plants alter their development and architecture in response to ambient temperature is crucial for breeding resilient crops. Here, we identify the quantitative trait locus qMULTIPLE INFLORESCENCE BRANCH 2 (qMIB2), which modulates inflorescence branching in response to high ambient temperature in tomato (Solanum lycopersicum). The non-functional mib2 allele may have been selected in large-fruited varieties to ensure larger and more uniform fruits under varying temperatures. MIB2 gene encodes a homolog of the Arabidopsis thaliana transcription factor SPATULA; its expression is induced in meristems at high temperature. MIB2 directly binds to the promoter of its downstream gene CONSTANS-Like1 (SlCOL1) by recognizing the conserved G-box motif to activate SlCOL1 expression in reproductive meristems. Overexpressing SlCOL1 rescue the reduced inflorescence branching of mib2, suggesting how the MIB2–SlCOL1 module helps tomato inflorescences adapt to high temperature. Our findings reveal the molecular mechanism underlying inflorescence thermomorphogenesis and provide a target for breeding climate-resilient crops.

A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1.

The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in the control of shoot branching as it integrates environmental and endogenous signals that influence axillary bud growth. Despite its remarkable activity as a growth inhibitor, the mechanisms by which BRC1 promotes bud dormancy are largely unknown. We determined the genome-wide BRC1 binding sites in vivo and combined these with transcriptomic data and gene co-expression analyses to identify bona fide BRC1 direct targets. Next, we integrated multi-omics data to infer the BRC1 gene regulatory network (GRN) and used graph theory techniques to find network motifs that control the GRN dynamics. We generated an open online tool to interrogate this network. A group of BRC1 target genes encoding transcription factors (BTFs) orchestrate this intricate transcriptional network enriched in abscisic acid-related components. Promoter::β-GLUCURONIDASE transgenic lines confirmed that BTFs are expressed in axillary buds. Transient co-expression assays and studies in planta using mutant lines validated the role of BTFs in modulating the GRN and promoting bud dormancy. This knowledge provides access to the developmental mechanisms that regulate shoot branching and helps identify candidate genes to use as tools to adapt plant architecture and crop production to ever-changing environmental conditions.

The trans-regulatory landscape of gene networks in plants.

The transcriptional effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative transcriptional effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how our characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply.

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Whereas several transcriptional regulators of nitrate signaling have been identified, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN7 (NLP7), additional regulators are expected to fine-tune this pivotal physiological response. Here, we characterized Arabidopsis NLP2 as a top-tier transcriptional regulator of the early nitrate response gene regulatory network. NLP2 interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, DNA-binding motif, and some target genes with NLP7. Genetic, genomic, and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multitiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

Ectopic Expression of Arabidopsis thaliana zDof1.3 in Tomato (Solanum lycopersicum L.) Is Associated with Improved Greenhouse Productivity and Enhanced Carbon and Nitrogen Use.

A large collection of transgenic tomato lines, each ectopically expressing a different Arabidopsis thaliana transcription factor, was screened for variants with alterations in leaf starch. Such lines may be affected in carbon partitioning, and in allocation to the sinks. We focused on ‘L4080’, which harbored an A. thaliana zDof (DNA-binding one zinc finger) isoform 1.3 (AtzDof1.3) gene, and which had a 2–4-fold higher starch-to-sucrose ratio in source leaves over the diel (p < 0.05). Our aim was to determine whether there were associated effects on productivity. L4080 plants were altered in nitrogen (N) and carbon (C) metabolism. The N-to-C ratio was higher in six-week-old L4080, and when treated with 1/10 N, L4080 growth was less inhibited compared to the wild-type and this was accompanied by faster root elongation (p < 0.05). The six-week-old L4080 acquired 42% more dry matter at 720 ppm CO2, compared to ambient CO2 (p < 0.05), while the wild-type (WT) remained unchanged. GC-MS-TOF data showed that L4080 source leaves were enriched in amino acids compared to the WT, and at 49 DPA, fruit had 25% greater mass, higher sucrose, and increased yield (25%; p < 0.05) compared to the WT. An Affymetrix cDNA array analysis suggested that only 0.39% of the 9000 cDNAs were altered by 1.5-fold (p < 0.01) in L4080 source leaves. 14C-labeling of fruit disks identified potential differences in 14-DPA fruit metabolism suggesting that post-transcriptional regulation was important. We conclude that AtzDof1.3 and the germplasm derived therefrom, should be investigated for their ‘climate-change adaptive’ potential.

Interaction between MdZAT10a-like and MdbHLH100 negatively regulates salt tolerance in apple (Malus domestica Borkh.)

Salinity is a harmful environmental factor that inhibits plant growth, and reduces the yield and quality of crops, including apple (Malus ×domestica Borkh.). In this study, we treated tissue-cultured wild-type apple ‘Royal Gala’ seedlings with different concentrations of NaCl. A zinc finger of Arabidopsis thaliana (ZAT) transcription factor, designated MdZAT10a-like, was screened by qRT-PCR analysis and shown to be significantly responsive to salt stress. Overexpression of MdZAT10a-like in apple callus reduced the expression of MdNHX1 and thereby enhanced the sensitivity of callus to salt stress. Yeast two-hybrid screening showed that MdbHLH100 was capable of interacting with MdZAT10a-like. The MdZAT10a-like–MdbHLH100 protein complex significantly inhibited the expression of the downstream gene MdNHX1. The present functional analyses of MdZAT10a-like provide valuable insights into the regulatory network of salt tolerance in apple.

Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein.

Transcription factors (TFs) play an important role in plant development; however, their role during viral infection largely remains unknown. The present study was designed to uncover the role transcription factors play in Cucumber mosaic virus (CMV) infection. During the screening of an Arabidopsis thaliana (Col-0) transcription factor library, using the CMV 2b protein as bait in the yeast two-hybrid system, the 2b protein interacted with Homeobox protein 27 (HB27). HB27 belongs to the zinc finger homeodomain family and is known to have a regulatory role in flower development, and responses to biotic and abiotic stress. The interaction between CMV 2b and HB27 proteins was further validated using in planta (bimolecular fluorescence complementation assay) and in vitro far-Western blotting (FWB) methods. In the bimolecular fluorescence complementation assay, these proteins reconstituted YFP fluorescence in the nucleus and the cytoplasmic region as small fluorescent dots. In FWB, positive interaction was detected using bait anti-MYC antibody on the target HB27-HA protein. During CMV infection, upregulation (~3-fold) of the HB27 transcript was observed at 14 days post-infection (dpi) in A. thaliana plants, and expression declined to the same as healthy plants at 21 dpi. To understand the role of the HB27 protein during CMV infection, virus accumulation was determined in HB27-overexpressing (HB27 OE) and knockout mutants. In HB27-overexpressing lines, infected plants developed mild symptoms, accumulating a lower virus titer at 21 dpi compared to wild-type plants. Additionally, knockout HB27 mutants had more severe symptoms and a higher viral accumulation than wild-type plants. These results indicate that HB27 plays an important role in the regulation of plant defense against plant virus infection.

A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model.

ABSTRACTCurrent seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection in vivo against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses.

Local DNA shape is a general principle of transcription factor binding specificity in Arabidopsis thaliana

Understanding gene expression will require understanding where regulatory factors bind genomic DNA. The frequently used sequence-based motifs of protein-DNA binding are not predictive, since a genome contains many more binding sites than are actually bound and transcription factors of the same family share similar DNA-binding motifs. Traditionally, these motifs only depict sequence but neglect DNA shape. Since shape may contribute non-linearly and combinational to binding, machine learning approaches ought to be able to better predict transcription factor binding. Here we show that a random forest machine learning approach, which incorporates the 3D-shape of DNA, enhances binding prediction for all 216 tested Arabidopsis thaliana transcription factors and improves the resolution of differential binding by transcription factor family members which share the same binding motif. We observed that DNA shape features were individually weighted for each transcription factor, even if they shared the same binding sequence.

A genome‐scale TF–DNA interaction network of transcriptional regulation of Arabidopsis primary and specialized metabolism

Plant metabolism is more complex relative to individual microbes. In single‐celled microbes, transcriptional regulation by single transcription factors (TFs) is sufficient to shift primary metabolism. Corresponding genome‐level transcriptional regulatory maps of metabolism reveal the underlying design principles responsible for these shifts as a model in which master regulators largely coordinate specific metabolic pathways. Plant primary and specialized metabolism occur within innumerable cell types, and their reactions shift depending on internal and external cues. Given the importance of plants and their metabolites in providing humanity with food, fiber, and medicine, we set out to develop a genome‐scale transcriptional regulatory map of Arabidopsis metabolic genes. A comprehensive set of protein–DNA interactions between Arabidopsis thaliana TFs and gene promoters in primary and specialized metabolic pathways were mapped. To demonstrate the utility of this resource, we identified and functionally validated regulators of the tricarboxylic acid (TCA) cycle. The resulting network suggests that plant metabolic design principles are distinct from those of microbes. Instead, metabolism appears to be transcriptionally coordinated via developmental‐ and stress‐conditional processes that can coordinate across primary and specialized metabolism. These data represent the most comprehensive resource of interactions between TFs and metabolic genes in plants.

PUCHI Regulates Giant Cell Morphology During Root-Knot Nematode Infection in Arabidopsis thaliana.

Parasitic root-knot nematodes transform the host’s vascular cells into permanent feeding giant cells (GCs) to withdraw nutrients from the host plants. GCs are multinucleated metabolically active cells with distinctive cell wall structures; however, the genetic regulation of GC formation is largely unknown. In this study, the functions of the Arabidopsis thaliana transcription factor PUCHI during GC development were investigated. PUCHI expression was shown to be induced in early developing galls, suggesting the importance of the PUCHI gene in gall formation. Despite the puchi mutant not differing significantly from the wild type in nematode invasion and reproduction rates, puchi GC cell walls appeared to be thicker and lobate when compared to the wild type, while the cell membrane sometimes formed invaginations. In three-dimensional (3D) reconstructions of puchi GCs, they appeared to be more irregularly shaped than those in the wild type, with noticeable cell-surface protrusions and folds. Interestingly, the loss-of-function mutant of 3-KETOACYL-COA SYNTHASE 1 showed GC morphology and cell wall defects similar to those of the puchi mutant, suggesting that PUCHI may regulate GC development via very long chain fatty acid synthesis.

Fiber Cell-Specific Expression of the VP16-Fused Ethylene Response Factor 41 Protein Increases Biomass Yield and Alters Lignin Composition.

Arabidopsis thaliana transcription factors belonging to the ERFIIId and ERFIIIe subclade (ERFIIId/e) of the APETALA 2/ethylene response factor (AP2/ERF) family enhance primary cell wall (PCW) formation. These transcription factors activate expression of genes encoding PCW-type cellulose synthase (CESA) subunits and other genes for PCW biosynthesis. In this study, we show that fiber-specific expression of ERF035-VP16 and ERF041-VP16, which are VP16-fused proteins of ERFIIId/e members, promote cell wall thickening in a wild-type background with a concomitant increase of alcohol insoluble residues (cell wall content) per fresh weight (FW) and monosaccharides related to the PCW without affecting plant growth. Furthermore, in the ERF041-VP16 lines, the total amount of lignin and the syringyl (S)/guaiacyl (G) ratio decreased, and the enzymatic saccharification yield of glucose from cellulose per fresh weight improved. In these lines, PCW-type CESA genes were upregulated and ferulate 5-hydropxylase1 (F5H1), which is necessary for production of the S unit lignin, was downregulated. In addition, various changes in the expression levels of transcription factors regulating secondary cell wall (SCW) formation were observed. In conclusion, fiber cell-specific ERF041-VP16 improves biomass yield, increases PCW components, and alters lignin composition and deposition and may be suitable for use in future molecular breeding programs of biomass crops.

Alterations in the silymarin metabolism in transgenic Silybum marianum cultured cells by the heterologous expression of the Arabidopsis thaliana V-myb myeloblastosis viral oncogene homolog transcription factor MYB12 and Cicer arietinum chalcone synthase

Silymarin (Sm), the main bioactive principle of Silybum marianum (L.) Gaernt. fruits, has strong antihepatotoxic properties and also exhibits antitumor and antiviral activity. This flavonolignan complex is synthesized from the flavonoid precursor taxifolin, which undergoes an oxidative coupling with the monolignol coniferyl alcohol. Attempts to produce Sm in plant cell suspensions have met with limited success, probably due to insufficient flux into the flavonoid branch pathway of Sm biosynthesis.In this study, it was attempted to increase Sm production in in vitro cultures of S. marianum by genetically engineering the flavonoid pathway. Using Agrobacterium tumefaciens strain C58C1, S. marianum cell suspension cultures were transformed either with the Arabidopsis thaliana transcription factor AtMYB12 (V-myb myeloblastosis viral oncogene homolog) (MYB) or with a chalcone synthase (CHS) gene from Cicer arietinum. A double transformant was also generated by introducing the CaCHS gene in AtMYB12-expressing cultures.The ectopic expression of AtMYB12 in cultures activated some of the genes involved in the flavonoid pathway, notably chalcone isomerase (CHI) and flavonoid 3′-hydroxylase (F3’H), whereas the secondary metabolites altered by the inserted gene were chlorogenic acid glycoside derivatives. Expression of CaCHS in the cultures led to the production of the flavanone naringenin (Ng), which accumulated as glycosides. The simultaneous expression of AtMYB12-CaCHS resulted in the production of lower amounts of chlorogenic derivatives but similar levels of Ng glycosides compared with the CaCHS lines. Both CaCHS lines and the double transformant could be maintained over prolonged subcultures without Ng production losses, confirming long-term and stable CaCHS transgene expression.In no case did the inserted genes have a discernable effect on Sm production compared with the untransformed cultures, neither under control culture conditions or in cultures elicited with MeJA.

A Gain-of-Function Screen Reveals Redundant ERF Transcription Factors Providing Opportunities for Resistance Breeding Toward the Vascular Fungal Pathogen Verticillium longisporum.

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.

Expression and regulatory asymmetry of retained Arabidopsis thaliana transcription factor genes derived from whole genome duplication

BackgroundTranscription factors (TFs) play a key role in regulating plant development and response to environmental stimuli. While most genes revert to single copy after whole genome duplication (WGD) event, transcription factors are retained at a significantly higher rate. Little is known about how TF duplicates have diverged in their expression and regulation, the answer to which may contribute to a better understanding of the elevated retention rate among TFs.ResultsHere we assessed what features may explain differences in the retention of TF duplicates and other genes using Arabidopsis thaliana as a model. We integrated 34 expression, sequence, and conservation features to build a linear model for predicting the extent of duplicate retention following WGD events among TFs and 19 groups of genes with other functions. We found that TFs was the least well predicted, demonstrating the features of TFs are substantially deviated from duplicate genes in other function groups. Consistent with this, the evolution of TF expression patterns and cis-regulatory cites favors the partitioning of ancestral states among the resulting duplicates: one “ancestral” TF duplicate retains most ancestral expression and cis-regulatory sites, while the “non-ancestral” duplicate is enriched for novel regulatory sites. By modeling the retention of ancestral expression and cis-regulatory states in duplicate pairs using a system of differential equations, we found that TF duplicate pairs in a partitioned state are preferentially maintained.ConclusionsThese TF duplicates with asymmetrically partitioned ancestral states are likely maintained because one copy retains ancestral functions while the other, at least in some cases, acquires novel cis-regulatory sites that may be important for novel, adaptive traits.

The Arabidopsis thaliana transcription factor MYB59 regulates calcium signalling during plant growth and stress response.

Transcription factor MYB59 is involved in plant growth and stress responses by acting as negative regulator of Ca signalling and homeostasis. The Arabidopsis thaliana transcription factor MYB59 is induced by cadmium (Cd) and plays a key role in the regulation of cell cycle progression and root elongation, but its mechanism of action is poorly understood. We investigated the expression of MYB59 and differences between wild-type plants, the myb59 mutant and MYB59-overexpressing lines (obtained by transformation in the mutant genotype) during plant growth and in response to various forms of stress. We also compared the transcriptomes of wild-type and myb59 mutant plants to determine putative MYB59 targets. The myb59 mutant has longer roots, smaller leaves and smaller cells than wild-type plants and responds differently to stress in germination assay. Transcriptomic analysis revealed the upregulation in the myb59 mutantof multiple genes involved in calcium (Ca) homeostasis and signalling, including those encoding calmodulin-like proteins and Ca transporters. Notably, MYB59 was strongly induced by Ca deficiency, and the myb59 mutant was characterized by higher levels of cytosolic Ca in root cells and showed a modest alteration of Ca transient frequency in guard cells, associated with the absence of Ca-induced stomatal closure. These results indicate that MYB59 negatively regulates Ca homeostasis and signalling during Ca deficiency, thus controlling plant growth and stress responses.

AtPAP1 Interacts With and Activates SmbHLH51, a Positive Regulator to Phenolic Acids Biosynthesis in Salvia miltiorrhiza.

Phenolic acids from Salvia miltiorrhiza have drawn considerable attention in recent years because of their remarkable pharmacological activities. We previously reported that Arabidopsis thaliana transcription factor production of anthocyanin pigment 1 (AtPAP1) has strong capability to promote the production of phenolic acids in S. miltiorrhiza. However, the responsible molecular mechanism is unclear. Here, we analyzed the transcriptome of transgenic S. miltiorrhiza that over-expressed AtPAP1. Transcriptome analysis revealed 4,152 genes that were differentially expressed due to ectopic AtPAP1 overexpression. SmbHLH51, a novel bHLH gene significantly up-regulated by constitutive expression of AtPAP1, was isolated from S. miltiorrhiza for detailed functional characterization. SmbHLH51 localizes in the nuclei and interacts with AtPAP1, indicating that they probably comprise a regulatory transcription complex. Enhanced or reduced expression of SmbHLH51 was achieved in S. miltiorrhiza by gain- or loss-of-function assays, respectively, revealing that SmbHLH51 is a positive transcriptional regulator of the pathway for phenolic acid biosynthesis. We propose that applying this functional genomics approach through the transcriptomic analyses is an efficient means for identifying novel genes involved in plant secondary metabolism.

The WOX11-LBD16 Pathway Promotes Pluripotency Acquisition in Callus Cells During De Novo Shoot Regeneration in Tissue Culture.

De novo shoot regeneration in tissue culture undergoes at least two phases. Explants are first cultured on auxin-rich callus-inducing medium (CIM) to produce a group of pluripotent cells termed callus; the callus is then transferred to cytokinin rich shoot-inducing medium (SIM) to promote the formation of shoot progenitor cells, from which adventitious shoots may differentiate. Here, we show that the Arabidopsis thaliana transcription factor gene LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) is involved in pluripotency acquisition in callus cells. LBD16, which is activated by WUSCHEL RELATED HOMEOBOX11 (WOX11), is specifically expressed in the newly formed callus on CIM and its expression decreases quickly when callus is moved to SIM. Blocking the WOX11-LBD16 pathway results in the loss of pluripotency in callus cultured on CIM, leading to shooting defects on SIM. Further analysis showed that LBD16 may function in the establishment of the root primordium-like identity in the newly formed callus, indicating that the root primordium-like identity is the cellular nature of pluripotency in callus cells. Additionally, LBD16 promotes cell division during callus initiation. Our study clarified that the WOX11-LBD16 pathway promotes pluripotency acquisition in callus cells.

Increased Phosphorylation of Ser-Gln Sites on SUPPRESSOR OF GAMMA RESPONSE1 Strengthens the DNA Damage Response in Arabidopsis thaliana.

The Arabidopsis thaliana transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) regulates hundreds of genes in response to DNA damage, and this results in the activation of cell cycle arrest, DNA repair, endoreduplication, and programmed cell death. However, it is not clear how this single transcription factor regulates each of these pathways. We previously reported that phosphorylation of five Ser-Gln (SQ) motifs in the C-terminal region of SOG1 are required to activate downstream pathways. In this study, we introduced Ser-to-Ala (AQ) substitutions in these five SQ motifs to progressively eliminate them and then we examined the effects on DNA damage responses. We found that all SQs are required for the full activation of SOG1 and that the expression level of most downstream genes changed incrementally depending on the number of phosphorylated SQ sites. Genes involved in DNA repair and cell cycle progression underwent stepwise activation and inhibition respectively as the number of phosphorylated SQ sites increased. Also, inhibition of DNA synthesis, programmed cell death, and cell differentiation were incrementally induced as the number of phosphorylated SQ sites increased. These results show that the extent of SQ phosphorylation in SOG1 regulates gene expression levels and determines the strength of DNA damage responses.