Arabidopsis Response Research Articles

MiCOL6, MiCOL7A and MiCOL7B isolated from mango regulate flowering and stress response in transgenic Arabidopsis.

The CONSTANS/CONSTANS-Like (CO/COL) family has been shown to play important roles in flowering, stress tolerance, fruit development and ripening in higher plants. In this study, three COL genes, MiCOL6, MiCOL7A and MiCOL7B, which each contain only one CCT domain, were isolated from mango (Mangifera indica), and their functions were investigated. MiCOL7A and MiCOL7B were expressed mainly at 20 days after flowering (DAF), and all three genes were highly expressed during the flowering induction period. The expression levels of the three genes were affected by light conditions, but only MiCOL6 exhibited a clear circadian rhythm. Overexpression of MiCOL6 promoted earlier flowering, while overexpression of MiCOL7A or MiCOL7B delayed flowering compared to that in the control lines of Arabidopsis thaliana under long-day (LD) and short-day (SD) conditions. Overexpressing MiCOL6, MiCOL7A or MiCOL7B in transgenic plants increased superoxide dismutase (SOD) and proline levels, decreased malondialdehyde (MAD) levels, and improved survival under drought and salt stress. In addition, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses showed that the MiCOL6, MiCOL7A and MiCOL7B proteins interact with several stress- and flower-related proteins. This work demonstrates the functions of MiCOL6, MiCOL7A and MiCOL7B and provides a foundation for further research on the role of mango COL genes in flowering regulation and the abiotic stress response.

Genome-wide association analysis reveals genes controlling an antagonistic effect of biotic and osmotic stress on Arabidopsis thaliana growth.

While the response of Arabidopsis thaliana to drought, herbivory or fungal infection has been well-examined, the consequences of exposure to a series of such (a)biotic stresses are not well studied. This work reports on the genetic mechanisms underlying the Arabidopsis response to single osmotic stress, and to combinatorial stress, either fungal infection using Botrytis cinerea or herbivory using Pieris rapae caterpillars followed by an osmotic stress treatment. Several small-effect genetic loci associated with rosette dry weight (DW), rosette water content (WC), and the projected rosette leaf area in response to combinatorial stress were mapped using univariate and multi-environment genome-wide association approaches. A single-nucleotide polymorphism (SNP) associated with DROUGHT-INDUCED 19 (DI19) was identified by both approaches, supporting its potential involvement in the response to combinatorial stress. Several SNPs were found to be in linkage disequilibrium with known stress-responsive genes such as PEROXIDASE 34 (PRX34), BASIC LEUCINE ZIPPER 25 (bZIP25), RESISTANCE METHYLATED GENE 1 (RMG1) and WHITE RUST RESISTANCE 4 (WRR4). An antagonistic effect between biotic and osmotic stress was found for prx34 and arf4 mutants, which suggests PRX34 and ARF4 play an important role in the response to the combinatorial stress.

Syntrichia ruralis: emerging model moss genome reveals a conserved and previously unknown regulator of desiccation in flowering plants.

Water scarcity, resulting from climate change, poses a significant threat to ecosystems. Syntrichia ruralis, a dryland desiccation-tolerant moss, provides valuable insights into survival of water-limited conditions. We sequenced the genome of S. ruralis, conducted transcriptomic analyses, and performed comparative genomic and transcriptomic analyses with existing genomes and transcriptomes, including with the close relative S. caninervis. We took a genetic approach to characterize the role of an S. ruralis transcription factor, identified in transcriptomic analyses, in Arabidopsis thaliana. The genome was assembled into 12 chromosomes encompassing 21 169 protein-coding genes. Comparative analysis revealed copy number and transcript abundance differences in known desiccation-associated gene families, and highlighted genome-level variation among species that may reflect adaptation to different habitats. A significant number of abscisic acid (ABA)-responsive genes were found to be negatively regulated by a MYB transcription factor (MYB55) that was upstream of the S. ruralis ortholog of ABA-insensitive 3 (ABI3). We determined that this conserved MYB transcription factor, uncharacterized in Arabidopsis, acts as a negative regulator of an ABA-dependent stress response in Arabidopsis. The new genomic resources from this emerging model moss offer novel insights into how plants regulate their responses to water deprivation.

Papiliotrema flavescens, a plant growth-promoting fungus, alters root system architecture and induces systemic resistance through its volatile organic compounds in Arabidopsis

The current trend in agricultural development is the establishment of sustainable agricultural systems. This involves utilizing and implementing eco-friendly biofertilizers and biocontrol agents as alternatives to conventional fertilizers and pesticides. A plant growth-promoting fungal strain, that could alter root system architecture and promote the growth of Arabidopsis seedlings in a non-contact manner by releasing volatile organic compounds (VOCs) was isolated in this study. 26S rDNA sequencing revealed that the strain was a yeast-like fungus, Papiliotrema flavescens. Analysis of plant growth-promoting traits revealed that the fungus could produce indole-3-acetic acid and ammonia and fix nitrogen. Transcriptome analysis in combination with inhibitor experiments revealed that P. flavescens VOCs triggered metabolic alterations, promoted auxin accumulation and distribution in the roots, and coordinated ethylene signaling, thus inhibiting primary root elongation and inducing lateral root formation in Arabidopsis. Additionally, transcriptome analysis and fungal infection experiments confirmed that pretreatment with P. flavescens stimulated the defense response of Arabidopsis to boost its resistance to the pathogenic fungus Botrytis cinerea. Solid-phase microextraction, which was followed by gas chromatography-mass spectrometry analysis, identified three VOCs (acetoin, naphthalene and indole) with significant plant growth-promoting attributes. Their roles were confirmed using further pharmacological experiments and upregulated expression of auxin- and ethylene-related genes. Our study serves as an essential reference for utilizing P. flavescens as a potential biological fertilizer and biocontrol agent.

A Glycosyl Hydrolase 5 Family Protein Is Essential for Virulence of Necrotrophic Fungi and Can Suppress Plant Immunity.

Phytopathogenic fungi normally secrete large amounts of CWDEs to enhance infection of plants. In this study, we identified and characterized a secreted glycosyl hydrolase 5 family member in Sclerotinia sclerotiorum (SsGH5, Sclerotinia sclerotiorum Glycosyl Hydrolase 5). SsGH5 was significantly upregulated during the early stages of infection. Knocking out SsGH5 did not affect the growth and acid production of S. sclerotiorum but resulted in decreased glucan utilization and significantly reduced virulence. In addition, Arabidopsis thaliana expressing SsGH5 became more susceptible to necrotrophic pathogens and basal immune responses were inhibited in these plants. Remarkably, the lost virulence of the ΔSsGH5 mutants was restored after inoculating onto SsGH5 transgenic Arabidopsis. In summary, these results highlight that S. sclerotiorum suppresses the immune responses of Arabidopsis through secreting SsGH5, and thus exerts full virulence for successful infection.

WRKY48 negatively regulates plant acclimation to a combination of high light and heat stress.

Plants growing under natural conditions experience high light (HL) intensities that are often accompanied by elevated temperatures. These conditions could affect photosynthesis, reduce yield, and negatively impact agricultural productivity. The combination of different abiotic challenges creates a new type of stress for plants by generating complex environmental conditions that often exceed the impact of their individual parts. Transcription factors (TFs) play a key role in integrating the different molecular signals generated by multiple stress conditions, orchestrating the acclimation response of plants to stress. In this study, we show that the TF WRKY48 negatively controls the acclimation of Arabidopsis thaliana plants to a combination of HL and heat stress (HL + HS), and its expression is attenuated by jasmonic acid under HL + HS conditions. Using comparative physiological and transcriptomic analyses between wild-type and wrky48 mutants, we further demonstrate that under control conditions, WRKY48 represses the expression of a set of transcripts that are specifically required for the acclimation of plants to HL + HS, hence its suppression during the HL + HS stress combination contributes to plant survival under these conditions. Accordingly, mutants that lack WRKY48 are more resistant to HL + HS, and transgenic plants that overexpress WRKY48 are more sensitive to it. Taken together, our findings reveal that WRKY48 is a negative regulator of the transcriptomic response of Arabidopsis to HL + HS and provide new insights into the complex regulatory networks of plant acclimation to stress combination.

Arabidopsis thaliana ubiquitin-associated protein 2 (AtUAP2) functions as an E4 ubiquitin factor and negatively modulates dehydration stress response.

E4, a ubiquitin (Ub) chain assembly factor and post-translational modification protein, plays a key role in the regulation of multiple cellular functions in plants during biotic or abiotic stress. We have more recently reported that E4 factor AtUAP1 is a negative regulator of the osmotic stress response and enhances the multi-Ub chain assembly of E3 ligase Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1). To further investigate the function of other E4 Ub factors in osmotic stress, we isolated AtUAP2, an AtUAP1 homolog, which interacted with AtRZF1, using pull-down assay and bimolecular fluorescence complementation analysis. AtUAP2, a Ub-associated motif-containing protein, interacts with oligo-Ub5, -Ub6, and -Ub7 chains. The yeast functional complementation experiment revealed that AtUAP2 functions as an E4 Ub factor. In addition, AtUAP2 is localized in the cytoplasm, different from AtUAP1. The activity of AtUAP2 was relatively strongly induced in the leaf tissue of AtUAP2 promoter-β-glucuronidase transgenic plants by abscisic acid, dehydration, and oxidative stress. atuap2 RNAi lines were more insensitive to osmotic stress condition than wild-type during the early growth of seedlings, whereas the AtUAP2-overexpressing line exhibited relatively more sensitive responses. Analyses of molecular and physiological experiments showed that AtUAP2 could negatively mediate the osmotic stress-induced signaling. Genetic studies showed that AtRZF1 mutation could suppress the dehydration-induced sensitive phenotype of the AtUAP2-overexpressing line, suggesting that AtRZF1 acts genetically downstream of AtUAP2 during osmotic stress. Taken together, our findings show that the AtRZF1-AtUAP2 complex may play important roles in the ubiquitination pathway, which controls the osmotic stress response in Arabidopsis.

Arabidopsis response to copper is mediated by density and root exudates: Evidence that plant density and toxic soils can shape plant communities.

Plants grown at high densities show increased tolerance to heavy metals for reasons that are not clear. A potential explanation is the release of citrate by plant roots, which binds metals and prevents uptake. Thus, pooled exudates at high plant densities might increase tolerance. We tested this exclusion facilitation hypothesis using mutants of Arabidopsis thaliana defective in citrate exudation. Wild type Arabidopsis and two allelic mutants for the Ferric Reductase Defective 3 (FRD3) gene were grown at four densities and watered with copper sulfate at four concentrations. Plants were harvested before bolting and dried. Shoot biomass was measured, and shoot material and soil were digested in nitric acid. Copper contents were determined by atomic absorption. In the highest-copper treatment, density-dependent reduction in toxicity was observed in the wild type but not in FRD3 mutants. For both mutants, copper concentrations per gram biomass were up to seven times higher than for wild type plants, depending on density and copper treatment. In all genotypes, total copper accumulation was greater at higher plant densities. Plant size variation increased with density and copper treatment because of heterogeneous distribution of copper throughout the soil. These results support the hypothesis that citrate exudation is responsible for density-dependent reductions in toxicity of metals. Density-dependent copper uptake and growth in contaminated soils underscores the importance of density in ecotoxicological testing. In soils with a heterogeneous distribution of contaminants, competition for nontoxic soil regions may drive size hierarchies and determine competitive outcomes.

Cytokinin signaling is involved in root hair elongation in response to phosphate starvation

ABSTRACT Root hair, single-celled tubular structures originating from the epidermis, plays a vital role in the uptake of nutrients from the soil by increasing the root surface area. Therefore, optimizing root hair growth is crucial for plants to survive in fluctuating environments. Root hair length is determined by the action of various plant hormones, among which the roles of auxin and ethylene have been extensively studied. However, evidence for the involvement of cytokinins has remained elusive. We recently reported that the cytokinin-activated B-type response regulators, ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12 directly upregulate the expression of ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4), which encodes a key transcription factor that controls root hair elongation. However, depending on the nutrient availability, it is unknown whether the ARR1/12–RSL4 pathway controls root hair elongation. This study shows that phosphate deficiency induced the expression of RSL4 and increased the root hair length through ARR1/12, though the transcript and protein levels of ARR1/12 did not change. These results indicate that cytokinins, together with other hormones, regulate root hair growth under phosphate starvation conditions.

Recent Advances in Photosynthesis, Plant Hormones and Applications in Plant Growth

Changes in the pigment and protein complexes involved in the light reactions of photosynthesis can be caused by environmental conditions, particularly those related to light quality and intensity during growth. The metabolome analysis of rice and soybean showed that dehydration stress increased the levels of galactinol and raffinose. One key method by which microorganisms control plant development has been proposed to be the generation of auxin or auxin precursors by bacteria associated with roots, or the manipulation of auxin production in plants. The effects of pathogenic and beneficial root-interacting fungi on the hormone content and auxin responsiveness of Arabidopsis, as determined by the auxin-inducible promoter system. Many developmental processes, like stem elongation, germination, vegetative growth, blooming, and reproduction, are regulated by GAs. Analyzing the metabolome of Several distinct defensive mechanisms function in different ways to prevent or lessen harm. Coeluting substances in the sample matrix have an effect on the analyte signals. They can impede or, less frequently, increase the analyte's ionization process, producing stronger or weaker signals, respectively. Furthermore, auxin, a different hormone present in plants, combines with cytokinins to prevent senescence, which, at least in its early stages, serves as a structured metabolic time and a tissue breakdown. Furthermore, synthetic amino acids with unique light-absorbing side chains may be used to add colors to such a new photosystem and rice showed that under circumstances of dehydration stress, levels of galactinol and raffinose were enhanced.

RNA N6-Methyladenosine Affects Copper-Induced Oxidative Stress Response in Arabidopsis thaliana.

Recently, post-transcriptional regulation of mRNA mediated by N6-methyladenosine (m6A) has been found to have profound effects on transcriptome regulation during plant responses to various abiotic stresses. However, whether this RNA modification can affect an oxidative stress response in plants has not been studied. To assess the role of m6A modifications during copper-induced oxidative stress responses, m6A-IP-seq was performed in Arabidopsis seedlings exposed to high levels of copper sulfate. This analysis revealed large-scale shifts in this modification on the transcripts most relevant for oxidative stress. This altered epitranscriptomic mark is known to influence transcript abundance and translation; therefore we scrutinized these possibilities. We found an increased abundance of copper-enriched m6A-containing transcripts. Similarly, we also found increased ribosome occupancy of copper-enriched m6A-containing transcripts, specifically those encoding proteins involved with stress responses relevant to oxidative stressors. Furthermore, the significance of the m6A epitranscriptome on plant oxidative stress tolerance was uncovered by assessing germination and seedling development of the mta (N6-methyladenosine RNA methyltransferase A mutant complemented with ABI3:MTA) mutant exposed to high copper treatment. These analyses suggested hypersensitivity of the mta mutant compared to the wild-type plants in response to copper-induced oxidative stress. Overall, our findings suggest an important role for m6A in the oxidative stress response of Arabidopsis.

Molecular evolution of methylesterase family genes and the BnMES34 is a positive regulator of Plasmodiophora brassicae stress response in Arabidopsis

Methylesterases (MES) are involved in hydrolysis of carboxylic esters, which have substantial roles in plant metabolic activities and defense mechanisms. This study aimed to comprehensively investigate Brassica napus BnMESs and characterize their role in response to Plasmodiophora brassicae stress. Forty-four BnMES members were identified and categorized into three groups based on their phylogenetic relationships and structural similarities. Through functional predictions in the promoter regions and analysis of RNA-Seq data, BnMES emerged as pivotal in growth, development, and stress responses to B. napus, particularly BnMES34, was strongly induced in response to P. brassicae infection. Gene Ontology analyses highlighted BnMES34's role in regulation of plant disease resistance responses. Furthermore, overexpression of BnMES34 in A. thaliana exhibited milder clubroot symptoms, and reduced disease indices, suggesting positive regulatory role of BnMES34 in plant's response to P. brassicae stress. Molecular docking and enzyme activity verification indicated that BnMES34 has the ability to generate salicylic acid via methyl salicylate, and further experimentally validated in vivo. This discovery indicates that the overexpression of BnMES34 in Arabidopsis confers resistance against clubroot disease. Overall, our research suggests that BnMES34 has a beneficial regulatory role in enhancing stress resistance to P. brassicae in B. napus.

The structure of B-ARR reveals the molecular basis of transcriptional activation by cytokinin.

The phytohormone cytokinin has various roles in plant development, including meristem maintenance, vascular differentiation, leaf senescence, and regeneration. Prior investigations have revealed that cytokinin acts via a phosphorelay similar to the two-component system by which bacteria sense and respond to external stimuli. The eventual targets of this phosphorelay are type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs), containing the conserved N-terminal receiver domain (RD), middle DNA binding domain (DBD), and C-terminal transactivation domain. While it has been established for two decades that the phosphoryl transfer from a specific histidyl residue in ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEINS (AHPs) to an aspartyl residue in the RD of B-ARRs results in a rapid transcriptional response to cytokinin, the underlying molecular basis remains unclear. In this work, we determine the crystal structures of the RD-DBD of ARR1 (ARR1RD-DBD) as well as the ARR1DBD-DNA complex from Arabidopsis. Analyses of the ARR1DBD-DNA complex have revealed the structural basis for sequence-specific recognition of the GAT trinucleotide by ARR1. In particular, comparing the ARR1RD-DBD and ARR1DBD-DNA structures reveals that unphosphorylated ARR1RD-DBD exists in a closed conformation with extensive contacts between the RD and DBD. In vitro and vivo functional assays have further suggested that phosphorylation of the RD weakens its interaction with DBD, subsequently permits the DNA binding capacity of DBD, and promotes the transcriptional activity of ARR1. Our findings thus provide mechanistic insights into phosphorelay activation of gene transcription in response to cytokinin.

Transcription factor MYB8 regulates iron deficiency stress response in Arabidopsis

Iron (Fe) is a crucial microelement for humans, animals, and plants. Insufficient Fe levels in plants impede growth and diminish photosynthesis, thus decreasing crop production. Notably, approximately one-third of the soil worldwide is alkaline and prone to Fe deficiency. Therefore, understanding the mechanisms underlying Fe absorption and transportation in plants can enhance Fe bioavailability in crops. In this study, the role of the transcription factor MYB8 in plant response to Fe deficiency in Arabidopsis was investigated via reverse genetics. Phenotype analysis revealed that the functional deletion mutant of MYB8 gene exhibited sensitivity to Fe deficiency stress, as indicated by shorter root length, lower chlorophyll content, and Fe concentration. Conversely, MYB8 overexpression strain showed a tolerant phenotype. Furthermore, qRT-PCR identified possible downstream MYB8-regulated genes. Moreover, MYB8 regulated the expression of iron-regulated transporter 1 (IRT1) by binding to the MYB binding sites motif (‘AACAAAC’) in its promoter.

FIONA1-mediated mRNA m6 A methylation regulates the response of Arabidopsis to salt stress.

N6 -methyladenosine (m6 A) is an mRNA modification widely found in eukaryotes and plays a crucial role in plant development and stress responses. FIONA1 (FIO1) is a recently identified m6 A methyltransferase that regulates Arabidopsis (Arabidopsis thaliana) floral transition; however, its role in stress response remains unknown. In this study, we demonstrate that FIO1-mediated m6 A methylation plays a vital role in salt stress response in Arabidopsis. The loss-of-function fio1 mutant was sensitive to salt stress. Importantly, the complementation lines expressing the wild-type FIO1 exhibited the wild-type phenotype, whereas the complementation lines expressing the mutant FIO1m , in which two critical amino acid residues essential for methyltransferase activity were mutated, did not recover the wild-type phenotype under salt stress, indicating that the salt sensitivity is associated with FIO1 methyltransferase activity. Furthermore, FIO1-mediated m6 A methylation regulated ROS production and affected the transcript level of several salt stress-responsive genes via modulating their mRNA stability in an m6 A-dependent manner in response to salt stress. Importantly, FIO1 is associated with salt stress response by specifically targeting and differentially modulating several salt stress-responsive genes compared with other m6 A writer. Collectively, our findings highlight the molecular mechanism of FIO1-mediated m6 A methylation in the salt stress adaptation.

Characterizing the role of PP2A B'' family subunits in mechanical stress response and plant development through calcium and ABA signaling in Arabidopsis thaliana.

Protein phosphatase 2AB'' (PP2A B'') family subunits have calcium-binding EF-hand motifs, facilitating interaction with PP2A substrates. In Arabidopsis thaliana, the PP2A B'' family subunits consist of six members, AtB''α-ε and FASS. These subunits can interact with a basic leucine zipper transcription factor, VIP1, and its close homologs. Mechanical stress triggers PP2A-mediated dephosphorylation of VIP1 and its close homologs, leading to nuclear localization and gene upregulation to alleviate touch-induced root bending and leaf damage. However, the physiological roles of PP2A B'' family subunits in the mechanical stress response in Arabidopsis remain unclear. This study aims to characterize such roles. A quadruple knockout mutant with T-DNA insertions in AtB''α, AtB''β, AtB''γ, and AtB''δ was generated. atb''αβγδ mutants exhibited no significant damage upon brushing or touch-induced root bending compared to the wild type. Transcriptome analysis showed a significant decrease in the expression of CYP707A3, a gene potentially targeted by VIP1 that regulates abscisic acid (ABA) catabolism, in the atb''αβγδ mutant compared to wild type leaves. However, other genes, including XTH23, EXLA1, and CYP707A1, also VIP1 targets, exhibited similar induction in both brushed atb''αβγδ mutants and wild type leaves. We observed an enrichment of the CAMTA motif, CGCG(C/T) in the promoters of genes showing downregulated expression levels in brushed atb''αβγδ leaves compared to brushed wild type leaves. These findings suggest that PP2A B'' family subunits exhibit functional redundancy in the VIP1-dependent pathway but influence CAMTA-dependent gene expression under mechanical stress. Under calcium-deficient and ABA-supplemented conditions, growth of atb''αβγδ seedlings was retarded when compared to wild type and single knockout mutants, atb''γ and atb''δ, indicating a crucial role in plant development by modulating calcium or ABA signaling.

Unfolded Protein Response in Arabidopsis.

The unfolded protein response (UPR) is a highly regulated signaling pathway that is largely conserved across eukaryotes. It is essential for cell homeostasis under environmental and physiological conditions that perturb the protein folding in the endoplasmic reticulum (ER). Arabidopsis is one of the outstanding multicellular model systems in which to investigate the UPR. Here, we described a protocol to induce the UPR in plants, specifically Arabidopsis, and to estimate their ability to cope with ER stress through the quantification of physiological parameters.

Heat Shock Factor A1s are required for phytochrome-interacting factor 4-mediated thermomorphogenesis in Arabidopsis.

Thermomorphogenesis and the heat shock (HS) response are distinct thermal responses in plants that are regulated by PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and HEAT SHOCK FACTOR A1s (HSFA1s), respectively. Little is known about whether these responses are interconnected and whether they are activated by similar mechanisms. An analysis of transcriptome dynamics in response to warm temperature (28°C) treatment revealed that 30 min of exposure activated the expression of a subset of HSFA1 target genes in Arabidopsis thaliana. Meanwhile, a loss-of-function HSFA1 quadruple mutant (hsfa1-cq) was insensitive to warm temperature-induced hypocotyl growth. In hsfa1-cq plants grown at 28°C, the protein and transcript levels of PIF4 were greatly reduced, and the circadian rhythm of many thermomorphogenesis-related genes (including PIF4) was disturbed. Additionally, the nuclear localization of HSFA1s and the binding of HSFA1d to the PIF4 promoter increased following warm temperature exposure, whereas PIF4 overexpression in hsfa1-cq partially rescued the altered warm temperature-induced hypocotyl growth of the mutant. Taken together, these results suggest that HSFA1s are required for PIF4 accumulation at a warm temperature, and they establish a central role for HSFA1s in regulating both thermomorphogenesis and HS responses in Arabidopsis.

Comparative functional analyses of PHR1, PHL1, and PHL4 transcription factors in regulating Arabidopsis responses to phosphate starvation.

To cope with phosphate (Pi) starvation, plants trigger an array of adaptive responses to sustain their growth and development. These responses are largely controlled at transcriptional levels. In Arabidopsis (Arabidopsis thaliana), PHOSPHATE RESPONSE 1 (PHR1) is a key regulator of plant physiological and transcriptional responses to Pi starvation. PHR1 belongs to a MYB-CC-type transcription factor family which contains 15 members. In this PHR1 family, PHR1/PHR1-like 1(PHL1) and PHL2/PHL3 form two distinct modules in regulating plant development and transcriptional responses to Pi starvation. PHL4 is the most closely related member to PHR1. Previously, using the phr1phl4 mutant, we showed that PHL4 is also involved in regulating plant Pi responses. However, the precise roles of PHL1 and PHL4 in regulating plant Pi responses and their functional relationships with PHR1 have not been clearly defined. In this work, we further used the phl1phl4 and phr1phl1phl4 mutants to perform comparative phenotypic and transcriptomic analyses with phr1, phr1phl1, and phr1phl4. The results showed that both PHL1 and PHL4 act redundantly and equally with PHR1 to regulate leaf senescence, Pi starvation induced-inhibition of primary root growth, and accumulation of anthocyanins in shoots. Unlike PHR1 and PHL1, however, the role of PHL4 in maintaining Pi homeostasis is negligible. In regulating transcriptional responses to Pi starvation at genomic levels, both PHL1 and PHL4 play minor roles when acts alone, however, they act synergistically with PHR1. In regulating Pi starvation-responsive genes, PHL4 also function less than PHL1 in terms of the number of the genes it regulates and the magnitude of gene transcription it affects. Furthermore, no synergistic interaction was found between PHL1 and PHL4 in regulating plant response to Pi starvation. Therefore, our results clarified the roles of PHL1 and PHL4 in regulating plant responses to Pi starvation. In addition, this work revealed a new function of these three transcription factors in regulating flowering time.

Modeling the Flowering Activation Motif during Vernalization in Legumes: A Case Study of M. trancatula.

In many plant species, flowering is promoted by the cold treatment or vernalization. The mechanism of vernalization-induced flowering has been extensively studied in Arabidopsis but remains largely unknown in legumes. The orthologs of the FLC gene, a major regulator of vernalization response in Arabidopsis, are absent or non-functional in the vernalization-sensitive legume species. Nevertheless, the legume integrator genes FT and SOC1 are involved in the transition of the vernalization signal to meristem identity genes, including PIM (AP1 ortholog). However, the regulatory contribution of these genes to PIM activation in legumes remains elusive. Here, we presented the theoretical and data-driven analyses of a feed-forward regulatory motif that includes a vernalization-responsive FT gene and several SOC1 genes, which independently activate PIM and thereby mediate floral transition. Our theoretical model showed that the multiple regulatory branches in this regulatory motif facilitated the elimination of no-sense signals and amplified useful signals from the upstream regulator. We further developed and analyzed four data-driven models of PIM activation in Medicago trancatula in vernalized and non-vernalized conditions in wild-type and fta1-1 mutants. The model with FTa1 providing both direct activation and indirect activation via three intermediate activators, SOC1a, SOC1b, and SOC1c, resulted in the most relevant PIM dynamics. In this model, the difference between regulatory inputs of SOC1 genes was nonessential. As a result, in the M. trancatula model, the cumulative action of SOC1a, SOC1b, and SOC1c was favored. Overall, in this study, we first presented the in silico analysis of vernalization-induced flowering in legumes. The considered vernalization network motif can be supplemented with additional regulatory branches as new experimental data become available.