student3Abstracts.txt

Distinct Clades of Protein Phosphatase 2A Regulatory B'/B56 Subunits Engage in Different Physiological Processes

Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'β clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'β being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.


Histone Deacetylase Complex 1 and histone 1 epigenetically moderate stress responsiveness of Arabidopsis thaliana seedlings

Early responses of plants to environmental stress factors prevent damage but can delay growth and development in fluctuating conditions. Optimising these trade-offs requires tunability of plant responsiveness to environmental signals. We have previously reported that Histone Deacetylase Complex 1 (HDC1), which interacts with multiple proteins in histone deacetylation complexes, regulates the stress responsiveness of Arabidopsis seedlings, but the underlying mechanism remained elusive. Here, we show that HDC1 attenuates transcriptome re-programming in salt-treated seedlings, and we identify two genes (LEA and MAF5) that inhibit seedling establishment under salt stress downstream of HDC1. HDC1 attenuates their transcriptional induction by salt via a dual mechanism involving H3K9/14 deacetylation and H3K27 trimethylation. The latter, but not the former, was also abolished in a triple knockout mutant of the linker histone H1, which partially mimics the hypersensitivity of the hdc1-1 mutant to salt stress. Although stress-induced H3K27me3 accumulation required both H1 and HDC1, it was not fully recovered by complementing hdc1-1 with a truncated, H1-binding competent HDC1 suggesting other players or independent inputs. The combined findings reveal a dual brake function of HDC1 via regulating both active and repressive epigenetic marks on stress-inducible genes. This natural 'anti-panic' device offers a molecular leaver to tune stress responsiveness in plants.


WRKY transcription factors and OBERON histone-binding proteins form complexes to balance plant growth and stress tolerance

WRKY transcription factors in plants are known to be able to mediate either transcriptional activation or repression, but the mechanism regulating their transcriptional activity is largely unclear. We found that group IId WRKY transcription factors interact with OBERON (OBE) proteins, forming redundant WRKY-OBE complexes in Arabidopsis thaliana. The coiled-coil domain of WRKY transcription factors binds to OBE proteins and is responsible for target gene selection and transcriptional repression. The PHD finger of OBE proteins binds to both histones and WRKY transcription factors. WRKY-OBE complexes repress the transcription of numerous stress-responsive genes and are required for maintaining normal plant growth. Several WRKY and OBE mutants show reduced plant size and increased drought tolerance, accompanied by increased expression of stress-responsive genes. Moreover, expression levels of most of these WRKY and OBE genes are reduced in response to drought stress, revealing a previously uncharacterized regulatory mechanism of the drought stress response. These results suggest that WRKY-OBE complexes repress transcription of stress-responsive genes, and thereby balance plant growth and stress tolerance.


Roles of the pepper JAZ protein CaJAZ1-03 and its interacting partner RING-type E3 ligase CaASRF1 in regulating ABA signaling and drought responses

Plants have developed various defense mechanisms against environmental stresses by regulating hormone signaling. Jasmonic acid (JA) is a major phytohormone associated with plant defense responses. JASMONATE ZIM-DOMAIN (JAZ) proteins play a regulatory role in repressing JA signaling, impacting plant responses to both biotic and abiotic stresses. Here, we isolated 7 JAZ genes in pepper and selected CA03g31030, a Capsicum annuum JAZ1-03 (CaJAZ1-03) gene, for further study based on its expression level in response to abiotic stresses. Through virus-induced gene silencing (VIGS) in pepper and overexpression in transgenic Arabidopsis plants, we established the functional role of CaJAZ1-03. Functional studies revealed that CaJAZ1-03 dampens abscisic acid (ABA) signaling and drought stress responses. The cell-free degradation assay showed faster degradation of CaJAZ1-03 in drought- or ABA-treated pepper leaves compared to healthy leaves. Conversely, CaJAZ1-03 was completely preserved under MG132 treatment, indicating that CaJAZ1-03 stability is modulated via the ubiquitin-26s proteasome pathway. We also found that the pepper RING-type E3 ligase CaASRF1 interacts with and ubiquitinates CaJAZ1-03. Additional cell-free degradation assays revealed a negative correlation between CaJAZ1-03 and CaASRF1 expression levels. Collectively, these findings suggest that CaJAZ1-03 negatively regulates ABA signaling and drought responses and that its protein stability is modulated by CaASRF1.


An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum

Excessive accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, which is an underlying cause of major crop losses and devastating human conditions. ER proteostasis surveillance is mediated by the conserved master regulator of the unfolded protein response (UPR), Inositol Requiring Enzyme 1 (IRE1), which determines cell fate by controlling pro-life and pro-death outcomes through as yet largely unknown mechanisms. Here we report that Arabidopsis IRE1 determines cell fate in ER stress by balancing the ubiquitin-proteasome system (UPS) and UPR through the plant-unique E3 ligase, PHOSPHATASE TYPE 2CA (PP2CA)-INTERACTING RING FINGER PROTEIN 1 (PIR1). Indeed, PIR1 loss leads to suppression of pro-death UPS and the lethal phenotype of an IRE1 loss-of-function mutant in unresolved ER stress in addition to activating pro-survival UPR. Specifically, in ER stress, PIR1 loss stabilizes ABI5, a basic leucine zipper (bZIP) transcription factor, that directly activates expression of the critical UPR regulator gene, bZIP60, triggering transcriptional cascades enhancing pro-survival UPR. Collectively, our results identify new cell fate effectors in plant ER stress by showing that IRE1's coordination of cell death and survival hinges on PIR1, a key pro-death component of the UPS, which controls ABI5, a pro-survival transcriptional activator of bZIP60.


Co-condensation with photoexcited cryptochromes facilitates MAC3A to positively control hypocotyl growth in Arabidopsis

Cryptochromes (CRYs) are blue light receptors that mediate plant photoresponses through regulating gene expressions. We recently reported that Arabidopsis CRY2 could form light-elicited liquid condensates to control RNA methylation. However, whether CRY2 condensation is involved in other gene expression-regulatory processes remains unclear. Here, we show that MOS4-associated complex subunits 3A and 3B (MAC3A/3B) are CRY-interacting proteins and assembled into nuclear CRY condensates. mac3a3b double mutants exhibit hypersensitive photoinhibition of hypocotyl elongation, suggesting that MAC3A/3B positively control hypocotyl growth. We demonstrate the noncanonical activity of MAC3A as a DNA binding protein that modulates transcription. Genome-wide mapping of MAC3A-binding sites reveals that blue light enhances the association of MAC3A with its DNA targets, which requires CRYs. Further evidence indicates that MAC3A and ELONGATED HYPOCOTYL 5 (HY5) occupy overlapping genomic regions and compete for the same targets. These results argue that photocondensation of CRYs fine-tunes light-responsive hypocotyl growth by balancing the opposed effects of HY5 and MAC3A.


The long non-coding RNA DANA2 positively regulates drought tolerance by recruiting ERF84 to promote JMJ29-mediated histone demethylation

Tens of thousands of long non-coding RNAs have been uncovered in plants, but few of them have been comprehensively studied for their biological function and molecular mechanism of their mode of action. Here, we show that the Arabidopsis long non-coding RNA DANA2 interacts with an AP2/ERF transcription factor ERF84 in the cell nucleus and then affects the transcription of JMJ29 that encodes a Jumonji C domain-containing histone H3K9 demethylase. Both RNA sequencing (RNA-seq) and genetic analyses demonstrate that DANA2 positively regulates drought stress responses through JMJ29. JMJ29 positively regulates the expression of ERF15 and GOLS2 by modulation of H3K9me2 demethylation. Accordingly, mutation of JMJ29 causes decreased ERF15 and GOLS2 expression, resulting in impaired drought tolerance, in agreement with drought-sensitive phenotypes of dana2 and erf84 mutants. Taken together, these results demonstrate that DANA2 is a positive regulator of drought response and works jointly with the transcriptional activator ERF84 to modulate JMJ29 expression in plant response to drought.


Genome-wide analysis of the MADS-box gene family in Lonicera japonica and a proposed floral organ identity model

BACKGROUND: Lonicera japonica Thunb. is widely used in traditional Chinese medicine. Medicinal L. japonica mainly consists of dried flower buds and partially opened flowers, thus flowers are an important quality indicator. MADS-box genes encode transcription factors that regulate flower development. However, little is known about these genes in L. japonica.RESULTS: In this study, 48 MADS-box genes were identified in L. japonica, including 20 Type-I genes (8 Mα, 2 Mβ, and 10 Mγ) and 28 Type-II genes (26 MIKCc and 2 MIKC*). The Type-I and Type-II genes differed significantly in gene structure, conserved domains, protein structure, chromosomal distribution, phylogenesis, and expression pattern. Type-I genes had a simpler gene structure, lacked the K domain, had low protein structure conservation, were tandemly distributed on the chromosomes, had more frequent lineage-specific duplications, and were expressed at low levels. In contrast, Type-II genes had a more complex gene structure; contained conserved M, I, K, and C domains; had highly conserved protein structure; and were expressed at high levels throughout the flowering period. Eleven floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in L. japonica. By integrating expression pattern and protein interaction data for these genes, we developed a possible model for floral organ identity determination.CONCLUSION: This study genome-widely identified and characterized the MADS-box gene family in L. japonica. Eleven floral homeotic MADS-box genes were identified and a possible model for floral organ identity determination was also developed. This study contributes to our understanding of the MADS-box gene family and its possible involvement in floral organ development in L. japonica.


RVE2, a new regulatory factor in jasmonic acid pathway, orchestrates resistance to Verticillium wilt

Verticillium dahliae, one of the most destructive fungal pathogens of several crops, challenges the sustainability of cotton productivity worldwide because very few widely-cultivated Upland cotton varieties are resistant to Verticillium wilt (VW). Here, we report that REVEILLE2 (RVE2), the Myb-like transcription factor, confers the novel function in resistance to VW by regulating the jasmonic acid (JA) pathway in cotton. RVE2 expression was essentially required for the activation of JA-mediated disease-resistance response. RVE2 physically interacted with TPL/TPRs and disturbed JAZ proteins to recruit TPL and TPR1 in NINJA-dependent manner, which regulated JA response by relieving inhibited-MYC2 activity. The MYC2 then bound to RVE2 promoter for the activation of its transcription, forming feedback loop. Interestingly, a unique truncated RVE2 widely existing in D-subgenome (GhRVE2D) of natural Upland cotton represses the ability of the MYC2 to activate GhRVE2A promoter but not GausRVE2 or GbRVE2. The result could partially explain why Gossypium barbadense popularly shows higher resistance than Gossypium hirsutum. Furthermore, disturbing the JA-signalling pathway resulted into the loss of RVE2-mediated disease-resistance in various plants (Arabidopsis, tobacco and cotton). RVE2 overexpression significantly enhanced the resistance to VW. Collectively, we conclude that RVE2, a new regulatory factor, plays a pivotal role in fine-tuning JA-signalling, which would improve our understanding the mechanisms underlying the resistance to VW.


Plant-microbe interactions: Plant-exuded myo-inositol attracts specific bacterial taxa

Plants exude a plethora of metabolites that transform the microbiome composition. Initiated from genome-wide association studies of either a plant or a bacterium, two new studies dissect the impact of plant-secreted myo-inositol on recruitment of certain bacterial taxa by Arabidopsis.


Genome-wide identification and expression pattern profiling of the ATP-binding cassette gene family in tea plant (Camelliasinensis)

The ATP-binding cassette (ABC) gene family is one of the largest and oldest protein families, consisting of ATP-driven transporters facilitating substrate transportation across cell membranes. However, little is known about the evolution and biological function of the ABC gene family in tea plants. In this study, we performed a genome-wide identification and expression analysis of genes encoding ABC transporter proteins in Camellia sinensis. Our analysis of 170 ABC genes revealed that CsABCs were unevenly distributed across 15 chromosomes, with an amino acid length ranging from 188 to 2489 aa, molecular weight ranging from 20.29 to 277.34 kDa, and an isoelectric point ranging from 4.89 to 10.63. Phylogenetic analysis showed that CsABCs were divided into eight subfamilies, among which the ABCG subfamily was the most abundant. Furthermore, the subcellular localization of CsABCs indicated that they were present in various organelles. Collinearity analysis between the tea plant and Arabidopsis thaliana genomes revealed that the CsABC genes were homologous to the AtABC genes. Large gene fragment duplication analysis identified ten gene pairs as tandem repeats, and interaction network analysis demonstrated that CsABCs interacted with various types of target genes, with protein interactions also occurring within the family. Tissue expression analysis indicated that CsABCs were highly expressed in roots, stems, and leaves and were easily induced by drought and cold stress. Moreover, qRT-PCR analysis of the relative expression level of the gene under drought and cold stress correlated with the sequencing results. Identifying ABC genes in tea plants lays a foundation for the classification and functional analysis of ABC family genes, which can facilitate molecular breeding and the development of new tea varieties.


3D organization of regulatory elements for transcriptional regulation in Arabidopsis

BACKGROUND: Although spatial organization of compartments and topologically associating domains at large scale is relatively well studied, the spatial organization of regulatory elements at fine scale is poorly understood in plants.RESULTS: Here we perform high-resolution chromatin interaction analysis using paired-end tag sequencing approach. We map chromatin interactions tethered with RNA polymerase II and associated with heterochromatic, transcriptionally active, and Polycomb-repressive histone modifications in Arabidopsis. Analysis of the regulatory repertoire shows that distal active cis-regulatory elements are linked to their target genes through long-range chromatin interactions with increased expression of the target genes, while poised cis-regulatory elements are linked to their target genes through long-range chromatin interactions with depressed expression of the target genes. Furthermore, we demonstrate that transcription factor MYC2 is critical for chromatin spatial organization, and propose that MYC2 occupancy and MYC2-mediated chromatin interactions coordinately facilitate transcription within the framework of 3D chromatin architecture. Analysis of functionally related gene-defined chromatin connectivity networks reveals that genes implicated in flowering-time control are functionally compartmentalized into separate subdomains via their spatial activity in the leaf or shoot apical meristem, linking active mark- or Polycomb-repressive mark-associated chromatin conformation to coordinated gene expression.CONCLUSION: The results reveal that the regulation of gene transcription in Arabidopsis is not only by linear juxtaposition, but also by long-range chromatin interactions. Our study uncovers the fine scale genome organization of Arabidopsis and the potential roles of such organization in orchestrating transcription and development.


The SOD7/DPA4-GIF1 module coordinates organ growth and iron uptake in Arabidopsis

Organ growth is controlled by both intrinsic genetic factors and external environmental signals. However, the molecular mechanisms that coordinate plant organ growth and nutrient supply remain largely unknown. We have previously reported that the B3 domain transcriptional repressor SOD7 (NGAL2) and its closest homologue DPA4 (NGAL3) act redundantly to limit organ and seed growth in Arabidopsis. Here we report that SOD7 represses the interaction between the transcriptional coactivator GRF-INTERACTING FACTOR 1 (GIF1) and growth-regulating factors (GRFs) by competitively interacting with GIF1, thereby limiting organ and seed growth. We further reveal that GIF1 physically interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), which acts as a central regulator of iron uptake and homeostasis. SOD7 can competitively repress the interaction of GIF1 with FIT to influence iron uptake and responses. The sod7-2 dpa4-3 mutant enhances the expression of genes involved in iron uptake and displays high iron accumulation. Genetic analyses support that GIF1 functions downstream of SOD7 to regulate organ and seed growth as well as iron uptake and responses. Thus, our findings define a previously unrecognized mechanism that the SOD7/DPA4-GIF1 module coordinates organ growth and iron uptake by targeting key regulators of growth and iron uptake.


KAI2 Can Do: Karrikin Receptor Function in Plant Development and Response to Abiotic and Biotic Factors

The α/β hydrolase KARRIKIN INSENSITIVE 2 (KAI2) functions as a receptor for a yet undiscovered phytohormone, provisionally termed KAI2 ligand (KL). In addition, it perceives karrikin, a butenolide compound found in the smoke of burnt plant material. KAI2-mediated signaling is involved in regulating seed germination and in shaping seedling and adult plant morphology, both above and below ground. It also governs responses to various abiotic stimuli and stresses and shapes biotic interactions. KAI2-mediated signaling is being linked to an elaborate cross-talk with other phytohormone pathways such as auxin, gibberellin, abscisic acid, ethylene and salicylic acid signaling, in addition to light and nutrient starvation signaling. Further connections will likely be revealed in the future. This article summarizes recent advances in unraveling the function of KAI2-mediated signaling and its interaction with other signaling pathways.


UV-B promotes flavonoid biosynthesis in Ginkgo biloba by inducing the GbHY5-GbMYB1-GbFLS module

Ginkgo biloba (ginkgo) leaves have medicinal value due to their high levels of secondary metabolites, such as flavonoids. We found that the flavonoid content in ginkgo leaves increases significantly at high altitudes (Qinghai-Tibet Plateau). Considering that high UV-B radiation is among the key environmental characteristics of the Qinghai-Tibet Plateau, we carried out simulated UV-B treatments on ginkgo seedlings and found that the flavonoid content of the leaves increased significantly following the treatments. Combined with results from our previous studies, we determined that the transcription factor GbHY5 may play a key role in responses to UV-B radiation. Overexpression of GbHY5 significantly promoted the accumulation of flavonoids in both ginkgo callus and Arabidopsis thaliana. Furthermore, yeast two-hybrid and real-time quantitative PCR showed that GbHY5 promoted the expression of GbMYB1 by interacting with GbMYB1 protein. Overexpression of GbMYB1 in ginkgo callus and A. thaliana also significantly promoted flavonoid biosynthesis. GbFLS encodes a key enzyme in flavonoid biosynthesis, and its promoter has binding elements of GbHY5 and GbMYB1. A dual-luciferase reporter assay indicated that while GbHY5 and GbMYB1 activated the expression of GbFLS individually, their co-expression achieved greater activation. Our analyses reveal the molecular mechanisms by which the UV-B-induced GbHY5-GbMYB1-GbFLS module promotes flavonoid biosynthesis in ginkgo, and they provide insight into the use of UV-B radiation to enhance the flavonoid content of ginkgo leaves.


Mechanosensitive ion channels MSL8, MSL9, and MSL10 have environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics in vitro

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N-terminal domains of MSL8, MSL9, and MSL10, three members of the MscS-like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N-termini of MSL channels are intrinsically disordered. We examined the N-terminus of MSL10 (MSL10N) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8N, MSL9N, and MSL10N have sharply differing propensities for self-assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment.


A GFP splicing reporter in a coilin mutant background reveals links between alternative splicing, siRNAs and coilin function in Arabidopsis thaliana

Coilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins (RNPs) including spliceosomal snRNPs. To study coilin function in plants, we conducted a genetic suppressor screen using a coilin (coi1) mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in older coi1 seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants of GFP pre-mRNA. In the coi1 suppressor screen, we identified suppressor mutations in WRAP53, a putative coilin-interacting protein; SMU2, a predicted splicing factor; and ZCH1, an incompletely characterized zinc finger protein. These suppressor mutations return the hyper-GFP fluorescence of young coi1 seedlings to the intermediate wild-type level. Additionally, coi1 zch1 mutants display more extensive GFP silencing and elevated levels of GFP siRNAs, suggesting the involvement of wild-type ZCH1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced the roles of coilin in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. The participation of coilin in these processes, at least some of which incorporate small RNAs, supports the hypothesis that coilin provides a chaperone for small RNA trafficking. Our study demonstrates the usefulness of the GFP splicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.


A root-knot nematode effector manipulates the rhizosphere microbiome for establishing parasitism relationship with hosts

INTRODUCTION: Root-knot nematode (RKN; Meloidogyne spp.) is one of the most infamous soilborne plant diseases, causing severe crop losses every year. Effector proteins secreted by RKNs play crucial roles during plant-nematode interaction. However, less is known about whether RKN effector proteins can impact the rhizosphere microbial environment.METHODS: In this study, we investigated the rhizosphere microbiome community of MiMIF-2 (a plant immunity-modulating effector) transgenic Arabidopsis thaliana with or without nematode infection using the Illumina high-throughput sequencing analysis.RESULTS AND DISCUSSION: The results showed that the bacterial species richness index increased, while the fungi species richness index decreased in M. incognita-infected MiMIF-2 transgenic A. thaliana plants. The relative abundance of genera such as Clitopilus, Komagataeibacter, Lactobacillus, Prevotella, Moritella, Vibrio, Escherichia-Shigella, and Pseudomonas was reduced in MiMIF-2 transgenic A. thaliana plants compared to wild type, but was significantly increased after inoculation with M. incognita. The Cluster of Orthologous Genes (COG) function classification analysis revealed a decrease in the relative abundance of defense mechanisms, secondary metabolite biosynthesis, transport, and nematode infection catabolism-related functions in MiMIF-2 lines compared to the wild type. These differences may be the reason for the increased susceptibility of MiMIF-2 transgenic A. thaliana to nematode infection. Our results provide a new insight into RKN effector proteins and their association with the microbial community, host, and plant pathogens, which will lead to the exploration of new innovative ideas for future biological control of RKNs.


The master growth regulator DELLA binding to histone H2A is essential for DELLA-mediated global transcription regulation

The DELLA genes, also known as 'Green Revolution' genes, encode conserved master growth regulators that control plant development in response to internal and environmental cues. Functioning as nuclear-localized transcription regulators, DELLAs modulate expression of target genes via direct protein-protein interaction of their carboxy-terminal GRAS domain with hundreds of transcription factors (TFs) and epigenetic regulators. However, the molecular mechanism of DELLA-mediated transcription reprogramming remains unclear. Here by characterizing new missense alleles of an Arabidopsis DELLA, repressor of ga1-3 (RGA), and co-immunoprecipitation assays, we show that RGA binds histone H2A via the PFYRE subdomain within its GRAS domain to form a TF-RGA-H2A complex at the target chromatin. Chromatin immunoprecipitation followed by sequencing analysis further shows that this activity is essential for RGA association with its target chromatin globally. Our results indicate that, although DELLAs are recruited to target promoters by binding to TFs via the LHR1 subdomain, DELLA-H2A interaction via the PFYRE subdomain is necessary to stabilize the TF-DELLA-H2A complex at the target chromatin. This study provides insights into the two distinct key modular functions in DELLA for its genome-wide transcription regulation in plants.


Plant Regulation Functions of Novel Phthalimide Compounds Based on AtPYL2

Novel agents contain the structure of phthalimide, which has antibacterial, insecticidal, and herbicidal activities. Recently, studies reported that these compounds can bind to plant hormone receptors and play important regulatory roles. In this study, the functions of agents were studied with in vitro and in vivo assays. The abscisic acid (ABA) receptor pyrabactin resistant-like 2 (PYL2) protein in Arabidopsis thaliana was expressed, purified, and crystallized; the analysis results of the crystal structure showed three AtPYL2 subunits in each asymmetric unit. The affinity of compounds Z1-Z11 to the AtPYL2 protein was tested by microscale thermophoresis (MST) and then verified by isothermal titration calorimetry (ITC). Furthermore, the binding pockets were found using molecular docking to verify the target relationships. Relevant in vivo assays for seed germination and a root growth assay were conducted, with the plant samples being treated with target compounds. The results show that the compounds Z3, Z5, and Z10 target AtPYL2 and that the dissociation constants for binding by MST were 3.59, 3.54, and 3.97 μmol/L, respectively, among them, and the molecular docking results showed that compounds Z3, Z5, and Z10 formed hydrophobic interactions with amino acid residues through hydrogen or halogen bonding. This highlights their potential as an ABA receptor protein agonist. On the other hand, in vivo, compounds Z3, Z5, and Z10 had different inhibitory effects on seed germination, with compound Z5 inhibiting the root growth of A. thaliana and compound Z10 affecting root growth. In conclusion, these compounds could regulate plant growth and could be further developed as new plant-regulating agents.


Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.


Moonlighting Arabidopsis molybdate transporter 2 family and GSH-complex formation facilitate molybdenum homeostasis

Molybdenum (Mo) as essential micronutrient for plants, acts as active component of molybdenum cofactor (Moco). Core metabolic processes like nitrate assimilation or abscisic-acid biosynthesis rely on Moco-dependent enzymes. Although a family of molybdate transport proteins (MOT1) is known to date in Arabidopsis, molybdate homeostasis remained unclear. Here we report a second family of molybdate transporters (MOT2) playing key roles in molybdate distribution and usage. KO phenotype-analyses, cellular and organ-specific localization, and connection to Moco-biosynthesis enzymes via protein-protein interaction suggest involvement in cellular import of molybdate in leaves and reproductive organs. Furthermore, we detected a glutathione-molybdate complex, which reveals how vacuolar storage is maintained. A putative Golgi S-adenosyl-methionine transport function was reported recently for the MOT2-family. Here, we propose a moonlighting function, since clear evidence of molybdate transport was found in a yeast-system. Our characterization of the MOT2-family and the detection of a glutathione-molybdate complex unveil the plant-wide way of molybdate.


BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling

Pathogen-associated molecular patterns (PAMPs) trigger plant innate immunity that acts as the first line of inducible defense against pathogen infection. A receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) functions as a signaling hub immediately downstream of multiple pattern recognition receptors (PRRs). It is known that PLANT U-BOX PROTEIN 25 (PUB25) and PUB26 ubiquitinate BIK1 and mediate BIK1 degradation. However, how BIK1 homeostasis is maintained is not fully understood. Here, we show that two closely related ubiquitin ligases, RING DOMAIN LIGASE 1 (RGLG1) and RGLG2, preferentially associate with the hypo-phosphorylated BIK1 and promote the association of BIK1 with the co-receptor for several PRRs, BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). PUB25 interacts with RGLG2 and mediates its degradation. In turn, RGLG2 represses the ubiquitin ligase activity of PUB25. RGLG1/2 suppress PUB25-mediated BIK1 degradation, promote BIK1 protein accumulation, and positively regulate immune signaling in a ubiquitin ligase activity-dependent manner. Our work reveals how BIK1 homeostasis is maintained by the interplay of different ubiquitin ligases.


Turnip crinkle virus-encoded suppressor of RNA silencing suppresses mRNA decay by interacting with Arabidopsis XRN4

Plant cells employ intricate defense mechanisms, including mRNA decay pathways, to counter viral infections. Among the RNA quality control (RQC) mechanisms, nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD) pathways play critical roles in recognizing and cleaving aberrant mRNA molecules. Turnip crinkle virus (TCV) is a plant virus that triggers mRNA decay pathways, but it has also evolved strategies to evade this antiviral defense. In this study, we investigated the activation of mRNA decay during TCV infection and its impact on TCV RNA accumulation. We found that TCV infection induced the upregulation of essential mRNA decay factors, indicating their involvement in antiviral defense and the capsid protein (CP) of TCV, a well-characterized viral suppressor of RNA silencing (VSR), also compromised the mRNA decay-based antiviral defense by targeting AtXRN4. This interference with mRNA decay was supported by the observation that TCV CP stabilized a reporter transcript with a long 3' untranslated region (UTR). Moreover, TCV CP suppressed the decay of known NMD target transcripts, further emphasizing its ability to modulate host RNA control mechanisms. Importantly, TCV CP physically interacted with AtXRN4, providing insight into the mechanism of viral interference with mRNA decay. Overall, our findings reveal an alternative strategy employed by TCV, wherein the viral coat protein suppresses the mRNA decay pathway to facilitate viral infection.


HISTONE DEACETYLASE 9 promotes hypocotyl-specific auxin response under shade

Vegetative shade causes an array of morphological changes in plants called shade avoidance syndrome, which includes hypocotyl and petiole elongation, leaf hyponasty, reduced leaf growth, early flowering and rapid senescence. Here, we show that loss-of-function mutations in HISTONE DEACETYLASE 9 (HDA9) attenuated the shade-induced hypocotyl elongation in Arabidopsis. However, the hda9 cotyledons and petioles under shade were not significantly different from those in wild-type, suggesting a specific function of HDA9 in hypocotyl elongation in response to shade. HDA9 expression levels were stable under shade and its protein was ubiquitously detected in cotyledon, hypocotyl and root. Organ-specific transcriptome analysis unraveled that shade induced a set of auxin-responsive genes, such as SMALL AUXIN UPREGULATED RNAs (SAURs) and AUXIN/INDOLE-3-ACETIC ACIDs (AUX/IAAs) and their induction was impaired in hda9-1 hypocotyls. In addition, HDA9 binding to loci of SAUR15/65, IAA5/6/19 and ACS4 was increased under shade. The genetic and organ-specific gene expression analyses further revealed that HDA9 may cooperate with PHYTOCHROME-INTERACTING FACTOR 4/7 in the regulation of shade-induced hypocotyl elongation. Furthermore, HDA9 and PIF7 proteins were found to interact together and thus it is suggested that PIF7 may recruit HDA9 to regulate the shade/auxin responsive genes in response to shade. Overall, our study unravels that HDA9 can work as one component of a hypocotyl-specific transcriptional regulatory machinery that activates the auxin response at the hypocotyl leading to the elongation of this organ under shade.


AXR1 modulates trichome morphogenesis through mediating ROP2 stability in Arabidopsis

Cell differentiation and morphogenesis are crucial for the establishment of diverse cell types and organs in multicellular organisms. Trichome cells offer an excellent paradigm for dissecting the regulatory mechanisms of plant cell differentiation and morphogenesis due to their unique growth characteristics. Here, we report the isolation of an Arabidopsis mutant, aberrantly branched trichome 3-1 (abt3-1), with a reduced trichome branching phenotype. Positional cloning and molecular complementation experiments confirmed that abt3-1 is a new mutant allele of Auxin resistant 1 (AXR1), which encodes the N-terminal half of ubiquitin-activating enzyme E1 and functions in auxin signaling pathway. Meanwhile, we found that transgenic plants expressing constitutively active version of ROP2 (CA-ROP2) caused a reduction of trichome branches, resembling that of abt3-1. ROP2 is a member of Rho GTPase of plants (ROP) family, serving as versatile signaling switches involved in a range of cellular and developmental processes. Our genetic and biochemical analyses showed AXR1 genetically interacted with ROP2 and mediated ROP2 protein stability. The loss of AXR1 aggravated the trichome defects of CA-ROP2 and induced the accumulation of steady-state ROP2. Consistently, elevated AXR1 expression levels suppressed ROP2 expression and partially rescued trichome branching defects in CA-ROP2 plants. Together, our results presented a new mutant allele of AXR1, uncovered the effects of AXR1 and ROP2 during trichome development, and revealed a pathway of ROP2-mediated regulation of plant cell morphogenesis in Arabidopsis.


Integration of eQTL and GWAS analysis uncovers a genetic regulation of natural ionomic variation in Arabidopsis

This study provided important insights into the genetic architecture of variations in A. thaliana leaf ionome in a cell-type-specific manner. The functional interpretation of traits associated variants by expression quantitative trait loci (eQTL) analysis is usually performed in bulk tissue samples. While the regulation of gene expression is context-dependent, such as cell-type-specific manner. In this study, we estimated cell-type abundances from 728 bulk tissue samples using single-cell RNA-sequencing dataset, and performed cis-eQTL mapping to identify cell-type-interaction eQTL (cis-eQTLs(ci)) in A. thaliana. Also, we performed Genome-wide association studies (GWAS) analyses for 999 accessions to identify the genetic basis of variations in A. thaliana leaf ionome. As a result, a total of 5,664 unique eQTL genes and 15,038 unique cis-eQTLs(ci) were significant. The majority (62.83%) of cis-eQTLs(ci) were cell-type-specific eQTLs. Using colocalization, we uncovered one interested gene AT2G25590 in Phloem cell, encoding a kind of plant Tudor-like protein with possible chromatin-associated functions, which colocalized with the most significant cis-eQTL(ci) of a Mo-related locus (Chr2:10,908,806:A:C; P = 3.27 × 10-27). Furthermore, we prioritized eight target genes associated with AT2G25590, which were previously reported in regulating the concentration of Mo element in A. thaliana. This study revealed the genetic regulation of ionomic variations and provided a foundation for further studies on molecular mechanisms of genetic variants controlling the A. thaliana ionome.


Asparagine-rich protein (NRP) mediates stress response by regulating biosynthesis of plant secondary metabolites in Arabidopsis

The plant-specific stress response protein NRP (asparagine-rich protein) is characterized by an asparagine-rich domain at its N-terminus and a conserved development and cell death (DCD) domain at its C-terminus. Previous transcriptional studies and phenotypic analyses have demonstrated the involvement of NRP in response to severe stress conditions, such as high salt and ER Endoplasmic reticulum-stress. We have recently identified distinct roles for NRP in biotic- and abiotic-stress signaling pathways, in which NRP interacts with different signaling proteins to change their subcellular localizations and stability. Here, to further explore the function of NRP, a transcriptome analysis was carried out on nrp1nrp2 knock-out lines at different life stages or under different growing conditions. The most significant changes in the transcriptome at both stages and conditions turned out to be the induction of the synthesis of secondary metabolites (SMs). Such an observation implicates that NRP is a general stress-responsive protein involved in various challenges faced by plants during their life cycle, which might involve a broad alteration in the distribution of SMs.


Arabidopsis Protein Phosphatase PIA1 Impairs Plant Drought Tolerance by Serving as a Common Negative Regulator in ABA Signaling Pathway

Reversible phosphorylation of proteins is a ubiquitous regulatory mechanism in vivo that can respond to external changes, and plays an extremely important role in cell signal transduction. Protein phosphatase 2C is the largest protein phosphatase family in higher plants. Recently, it has been found that some clade A members can negatively regulate ABA signaling pathways. However, the functions of several subgroups of Arabidopsis PP2C other than clade A have not been reported, and whether other members of the PP2C family also participate in the regulation of ABA signaling pathways remains to be studied. In this study, based on the previous screening and identification work of PP2C involved in the ABA pathway, the clade F member PIA1 encoding a gene of the PP2C family, which was down-regulated after ABA treatment during the screening, was selected as the target. Overexpression of PIA1 significantly down-regulated the expression of ABA marker gene RD29A in Arabidopsis protoplasts, and ABA-responsive elements have been found in the cis-regulatory elements of PIA1 by promoter analysis. When compared to Col-0, transgenic plants overexpressing PIA1 were less sensitive to ABA, whereas pia1 showed the opposite trait in seed germination, root growth, and stomatal opening experiments. Under drought stress, SOD, POD, CAT, and APX activities of PIA1 overexpression lines were lower than Col-0 and pia1, while the content of H2O2 was higher, leading to its lowest survival rate in test plants, which were consistent with the significant inhibition of the expression of ABA-dependent stress-responsive genes RD29B, ABI5, ABF3, and ABF4 in the PIA1 transgenic background after ABA treatment. Using yeast two-hybrid and luciferase complementation assays, PIA1 was found to interact with multiple ABA key signaling elements, including 2 RCARs and 6 SnRK2s. Our results indicate that PIA1 may reduce plant drought tolerance by functioning as a common negative regulator involved in ABA signaling pathway.


MBD3 Regulates Male Germ Cell Division and Sperm Fertility in Arabidopsis thaliana

DNA methylation plays important roles through the methyl-CpG-binding domain (MBD) to realize epigenetic modifications. Thirteen AtMBD proteins have been identified from the Arabidopsis thaliana genome, but the functions of some members are unclear. AtMBD3 was found to be highly expressed in pollen and seeds and it preferably binds methylated CG, CHG, and unmethylated DNA sequences. Then, two mutant alleles at the AtMBD3 locus were obtained in order to further explore its function using CRISPR/Cas9. When compared with 92.17% mature pollen production in the wild type, significantly lower percentages of 84.31% and 78.91% were observed in the mbd3-1 and mbd3-2 mutants, respectively. About 16-21% of pollen from the mbd3 mutants suffered a collapse in reproductive transmission, whereas the other pollen was found to be normal. After pollination, about 16% and 24% of mbd3-1 and mbd3-2 mutant seeds underwent early or late abortion, respectively. Among all the late abortion seeds in mbd3-2 plants, 25% of the abnormal seeds were at the globular stage, 31.25% were at the transition stage, and 43.75% were at the heart stage. A transcriptome analysis of the seeds found 950 upregulated genes and 1128 downregulated genes between wild type and mbd3-2 mutants. Some transcriptional factors involved in embryo development were selected to be expressed, and we found significant differences between wild type and mbd3 mutants, such as WOXs, CUC1, AIB4, and RGL3. Furthermore, we found a gene that is specifically expressed in pollen, named PBL6. PBL6 was found to directly interact with AtMBD3. Our results provide insights into the function of AtMBD3 in plants, especially in sperm fertility.


Comprehensive Characterization of B-Box Zinc Finger Genes in Citrullus lanatus and Their Response to Hormone and Abiotic Stresses

Plant B-BOX (BBX) zinc finger transcription factors play crucial roles in growth and development and the stress response. Although the BBX family has been characterized in various plants, systematic analysis in watermelon is still lacking. In this study, 25 watermelon ClBBX genes were identified. ClBBXs were grouped into five clades (Clade I, II, III, IV, and V) based on their conserved domains and phylogenetic relationships. Most of the ClBBXs (84%) might be localized in the nuclei or cytoplasm. The classification of ClBBXs was consistent with their gene structures. They were unevenly distributed in nine chromosomes except for Chr4 and Chr10, with the largest number of six members in Chr2. Segmental duplications were the major factor in ClBBX family expansion. Some BBXs of watermelon and Arabidopsis evolved from a common ancestor. In total, 254 hormonal and stress-responsive cis elements were discovered in ClBBX promoters. ClBBXs were differentially expressed in tissues, and the expression levels of ClBBX15 and 16 were higher in aboveground tissues than in roots, while the patterns of ClBBX21a, 21b, 21c, 28 and 30b were the opposite. With salicylic acid, methyl jasmonate and salt stress conditions, 17, 18 and 18 ClBBXs exhibited significant expression changes, respectively. In addition, many ClBBXs, including ClBBX29b, 30a and 30b, were also responsive to cold and osmotic stress. In summary, the simultaneous response of multiple ClBBXs to hormonal or abiotic stress suggests that they may have functional interactions in the stress hormone network. Clarifying the roles of key ClBBXs in transcriptional regulation and mediating protein interactions will be an important task. Our comprehensive characterization of the watermelon ClBBX family provides vital clues for the in-depth analysis of their biological functions in stress and hormone signaling pathways.


Genome-Wide Analyses of SlFWL Family Genes and Their Expression Profiles under Cold, Heat, Salt and Drought Stress in Tomato

PLAC8 is a cysteine-rich protein that serves as a central mediator of tumor evolution in mammals. PLAC8 motif-containing proteins widely distribute in fungi, algae, higher plants and animals that have been described to be implicated in fruit size, cell number and the transport of heavy metals such as cadmium or zinc. In tomatoes, FW2.2 is a PLAC8 motif-containing gene that negatively controls fruit size by regulating cell division and expansion in the carpel ovary during fruit development. However, despite FW2.2, other FWL (FW2.2-Like) genes in tomatoes have not been investigated. In this study, we identified the 21 SlFWL genes, including FW2.2, examined their expression profiles under various abiotic adversity-related conditions. The SlFWL gene structures and motif compositions are conserved, indicating that tomato SlFWL genes may have similar roles. Cis-acting element analysis revealed that the SlFWL genes may participate in light and abiotic stress responses, and they also interacted with a variety of phytohormone-responsive proteins and plant development elements. Phylogenetic analyses were performed on five additional plant species, including Arabidopsis, pepper, soybean, rice and maize, these genes were classified into five subfamilies. Based on the results of collinearity analyses, the SlFWL genes have a tighter homologous evolutionary relationship with soybean, and these orthologous FWL gene pairs might have the common ancestor. Expression profiling of SlFWL genes show that they were all responsive to abiotic stresses, each subgroup of genes exhibited a different expression trend. Our findings provide a strong foundation for investigating the function and abiotic stress responses of the SlFWL family genes.


The Modulation of Sucrose Nonfermenting 1-Related Protein Kinase 2.6 State by Persulfidation and Phosphorylation: Insights from Molecular Dynamics Simulations

SnRK2.6 (SUCROSE NONFERMENTING 1-RELATED PROTEIN KINASE2.6) has been characterized as a molecular switch for the intracellular abscisic acid (ABA) signal-transduction pathway. Normally, SnRK2.6 is kept in an "off" state, forming a binary complex with protein phosphatase type 2Cs (PP2Cs). Upon stressful conditions, SnRK2.6 turns into an "on" state by its release from PP2Cs and then phosphorylation at Ser175. However, how the "on" and "off" states for SnRK2.6 are fine-tuned, thereby controlling the initiation and braking processes of ABA signaling, is still largely unclear. SnRK2.6 activity was tightly regulated through protein post-translational modifications (PTM), such as persulfidation and phosphorylation. Taking advantage of molecular dynamics simulations, our results showed that Cys131/137 persulfidation on SnRK2.6 induces destabilized binding and weakened interactions between SnRK2.6 and HAB1 (HYPERSENSITIVE TO ABA1), an important PP2C family protein. This unfavorable effect on the association of the SnRK2.6-HAB1 complex suggests that persulfidation functions are a positive regulator of ABA signaling initiation. In addition, Ser267 phosphorylation in persulfidated SnRK2.6 renders a stable physical association between SnRK2.6 and HAB1, a key characterization for SnRK2.6 inhibition. Rather than Ser175, HAB1 cannot dephosphorylate Ser267 in SnRK2.6, which implies that the retained phosphorylation status of Ser267 could ensure that the activated SnRK2.6 reforms the binary complex to cease ABA signaling. Taken together, our findings expand current knowledge concerning the regulation of persulfidation and phosphorylation on the state transition of SnRK2.6 and provide insights into the fine-tuned mechanism of ABA signaling.


Arabidopsis Transcription Regulatory Factor Domain/Domain Interaction Analysis Tool-Liquid/Liquid Phase Separation, Oligomerization, GO Analysis: A Toolkit for Interaction Data-Based Domain Analysis

Although a large number of databases are available for regulatory elements, a bottleneck has been created by the lack of bioinformatics tools to predict the interaction modes of regulatory elements. To reduce this gap, we developed the Arabidopsis Transcription Regulatory Factor Domain/Domain Interaction Analysis Tool-liquid/liquid phase separation (LLPS), oligomerization, GO analysis (ART FOUNDATION-LOG), a useful toolkit for protein-nucleic acid interaction (PNI) and protein-protein interaction (PPI) analysis based on domain-domain interactions (DDIs). LLPS, protein oligomerization, the structural properties of protein domains, and protein modifications are major components in the orchestration of the spatiotemporal dynamics of PPIs and PNIs. Our goal is to integrate PPI/PNI information into the development of a prediction model for identifying important genetic variants in peaches. Our program unified interdatabase relational keys based on protein domains to facilitate inference from the model species. A key advantage of this program lies in the integrated information of related features, such as protein oligomerization, LOG analysis, structural characterizations of domains (e.g., domain linkers, intrinsically disordered regions, DDIs, domain-motif (peptide) interactions, beta sheets, and transmembrane helices), and post-translational modification. We provided simple tests to demonstrate how to use this program, which can be applied to other eukaryotic organisms.


Nitric Oxide and Globin Glb1 Regulate Fusarium oxysporum Infection of Arabidopsis thaliana

Plants continuously interact with fungi, some of which, such as Fusarium oxysporum, are lethal, leading to reduced crop yields. Recently, nitric oxide (NO) has been found to play a regulatory role in plant responses to F. oxysporum, although the underlying mechanisms involved are poorly understood. In this study, we show that Arabidopsis mutants with altered levels of phytoglobin 1 (Glb1) have a higher survival rate than wild type (WT) after infection with F. oxysporum, although all the genotypes analyzed exhibited a similar fungal burden. None of the defense responses that were analyzed in Glb1 lines, such as phenols, iron metabolism, peroxidase activity, or reactive oxygen species (ROS) production, appear to explain their higher survival rates. However, the early induction of the PR genes may be one of the reasons for the observed survival rate of Glb1 lines infected with F. oxysporum. Furthermore, while PR1 expression was induced in Glb1 lines very early on the response to F. oxysporum, this induction was not observed in WT plants.


Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1

Arabidopsis thaliana cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to 3E ↔ [E2]⧧ ↔ 1E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.


Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.


One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells

BACKGROUND: A commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters.RESULTS: As a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable.CONCLUSIONS: We conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.


Immune activation during Pseudomonas infection causes local cell wall remodeling and alters AGP accumulation

The plant cell boundary generally comprises constituents of the primary and secondary cell wall (CW) that are deposited sequentially during development. Although it is known that the CW acts as a barrier against phytopathogens and undergoes modifications to limit their invasion, the extent, sequence, and requirements of the pathogen-induced modifications of the CW components are still largely unknown, especially at the level of the polysaccharide fraction. To address this significant knowledge gap, we adopted the compatible Pseudomonas syringae-Arabidopsis thaliana system. We found that, despite systemic signaling actuation, Pseudomonas infection leads only to local CW modifications. Furthermore, by utilizing a combination of CW and immune signaling-deficient mutants infected with virulent or non-virulent bacteria, we demonstrated that the pathogen-induced changes in CW polysaccharides depend on the combination of pathogen virulence and the host's ability to mount an immune response. This results in a pathogen-driven accumulation of CW hexoses, such as galactose, and an immune signaling-dependent increase in CW pentoses, mainly arabinose, and xylose. Our analyses of CW changes during disease progression also revealed a distinct spatiotemporal pattern of arabinogalactan protein (AGP) deposition and significant modifications of rhamnogalacturonan sidechains. Furthermore, genetic analyses demonstrated a critical role of AGPs, specifically of the Arabinoxylan Pectin Arabinogalactan Protein1, in limiting pathogen growth. Collectively, our results provide evidence for the actuation of significant remodeling of CW polysaccharides in a compatible host-pathogen interaction, and, by identifying AGPs as critical elements of the CW in plant defense, they pinpoint opportunities to improve plants against diverse pathogens.


Understanding the salt overly sensitive pathway in Prunus: Identification and characterization of NHX, CIPK, and CBL genes

Salinity is a major abiotic stress factor that can significantly impact crop growth, and productivity. In response to salt stress, the plant Salt Overly Sensitive (SOS) signaling pathway regulates the homeostasis of intracellular sodium ion concentration. The SOS1, SOS2, and SOS3 genes play critical roles in the SOS pathway, which belongs to the members of Na+ /H+ exchanger (NHX), CBL-interacting protein kinase (CIPK), and calcineurin B-like (CBL) gene families, respectively. In this study, we performed genome-wide identifications and phylogenetic analyses of NHX, CIPK, and CBL genes in six Rosaceae species: Prunus persica, Prunus dulcis, Prunus mume, Prunus armeniaca, Pyrus ussuriensis × Pyrus communis, and Rosa chinensis. NHX, CIPK, and CBL genes of Arabidopsis thaliana were used as controls for phylogenetic analyses. Our analysis revealed the lineage-specific and adaptive evolutions of Rosaceae genes. Our observations indicated the existence of two primary classes of CIPK genes: those that are intron-rich and those that are intron-less. Intron-rich CIPKs in Rosaceae and Arabidopsis can be traced back to algae CIPKs and CIPKs found in early plants, suggesting that intron-less CIPKs evolved from their intron-rich counterparts. This study identified one gene for each member of the SOS signaling pathway in P. persica: PpSOS1, PpSOS2, and PpSOS3. Gene expression analyses indicated that all three genes of P. persica were expressed in roots and leaves. Yeast two-hybrid-based protein-protein interaction analyses revealed a direct interaction between PpSOS3 and PpSOS2; and between PpSOS2 and PpSOS1C-terminus region. Our findings indicate that the SOS signaling pathway is highly conserved in P. persica.