Heat-induced SUMOylation differentially affects bacterial effectors in plant cells.

Bacterial pathogens deliver effectors into host cells to suppress immunity. How host cells target these effectors is critical in pathogen-host interactions. SUMOylation, an important type of posttranslational modification in eukaryotic cells, plays a critical role in immunity, but its effect on bacterial effectors remains unclear in plant cells. In this study, using bioinformatic and biochemical approaches, we found that at least 16 effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 are SUMOylated by the enzyme cascade from Arabidopsis thaliana. Mutation of SUMOylation sites on the effector HopB1 enhances its function in the induction of plant cell death via stability attenuation of a plant receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1. By contrast, SUMOylation is essential for the function of another effector, HopG1, in the inhibition of mitochondria activity and jasmonic acid signaling. SUMOylation of both HopB1 and HopG1 is increased by heat treatment, and this modification modulates the functions of these 2 effectors in different ways in the regulation of plant survival rates, gene expression, and bacterial infection under high temperatures. Therefore, the current work on the SUMOylation of effectors in plant cells improves our understanding of the function of dynamic protein modifications in plant-pathogen interactions in response to environmental conditions.

S-acylation of a non-secreted peptide controls plant immunity via secreted-peptide signal activation.

Small peptides modulate multiple processes in plant cells, but their regulation by post-translational modification remains unclear. ROT4 (ROTUNDIFOLIA4) belongs to a family of Arabidopsis non-secreted small peptides, but knowledge on its molecular function and how it is regulated is limited. Here, we find that ROT4 is S-acylated in plant cells. S-acylation is an important form of protein lipidation, yet so far it has not been reported to regulate small peptides in plants. We show that this modification is essential for the plasma membrane association of ROT4. Overexpression of S-acylated ROT4 results in a dramatic increase in immune gene expression. S-acylation of ROT4 enhances its interaction with BSK5 (BRASSINOSTEROID-SIGNALING KINASE 5) to block the association between BSK5 and PEPR1 (PEP RECEPTOR1), a receptor kinase for secreted plant elicitor peptides (PEPs), thereby activating immune signaling. Phenotype analysis indicates that S-acylation is necessary for ROT4 functions in pathogen resistance, PEP response, and the regulation of development. Collectively, our work reveals an important role for S-acylation in the cross-talk of non-secreted and secreted peptide signaling in plant immunity.

Haplotype-resolved genomes of octoploid species in Phyllanthaceae family reveal a critical role for polyploidization and hybridization in speciation.

The Phyllanthaceae family comprises a diverse range of plants with medicinal, edible, and ornamental value, extensively cultivated worldwide. Polyploid species commonly occur in Phyllanthaceae. Due to the rather complex genomes and evolutionary histories, their speciation process has been still lacking in research. In this study, we generated chromosome-scale haplotype-resolved genomes of two octoploid species (Phyllanthus emblica and Sauropus spatulifolius) in Phyllanthaceae family. Combined with our previously reported one tetraploid (Sauropus androgynus) and one diploid species (Phyllanthus cochinchinensis) from the same family, we explored their speciation history. The three polyploid species were all identified as allopolyploids with subgenome A/B. Each of their two distinct subgenome groups from various species was uncovered to independently share a common diploid ancestor (Ancestor-AA and Ancestor-BB). Via different evolutionary routes, comprising various scenarios of bifurcating divergence, allopolyploidization (hybrid polyploidization), and autopolyploidization, they finally evolved to the current tetraploid S. androgynus, and octoploid S. spatulifolius and P. emblica, respectively. We further discuss the variations in copy number of alleles and the potential impacts within the two octoploids. In addition, we also investigated the fluctuation of metabolites with medical values and identified the key factor in its biosynthesis process in octoploids species. Our study reconstructed the evolutionary history of these Phyllanthaceae species, highlighting the critical roles of polyploidization and hybridization in their speciation processes. The high-quality genomes of the two octoploid species provide valuable genomic resources for further research of evolution and functional genomics.

A diRNA-protein scaffold module mediates SMC5/6 recruitment in plant DNA repair.

In eukaryotes, the STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 (SMC5/6) complex is critical to maintaining chromosomal structures around double-strand breaks (DSBs) in DNA damage repair. However, the recruitment mechanism of this conserved complex at DSBs remains unclear. In this study, using Arabidopsis thaliana as a model, we found that SMC5/6 localization at DSBs is dependent on the protein scaffold containing INVOLVED IN DE NOVO 2 (IDN2), CELL DIVISION CYCLE 5 (CDC5), and ALTERATION/DEFICIENCY IN ACTIVATION 2B (ADA2b), whose recruitment is further mediated by DNA-damage-induced RNAs (diRNAs) generated from DNA regions around DSBs. The physical interactions of protein components including SMC5-ADA2b, ADA2b-CDC5, and CDC5-IDN2 result in formation of the protein scaffold. Further analysis indicated that the DSB localization of IDN2 requires its RNA-binding activity and ARGONAUTE 2 (AGO2), indicating a role for the AGO2-diRNA complex in this process. Given that most of the components in the scaffold are conserved, the mechanism presented here, which connects SMC5/6 recruitment and small RNAs, will improve our understanding of DNA repair mechanisms in eukaryotes.

The chromosome-scale assembly of the Salvia rosmarinus genome provides insight into carnosic acid biosynthesis.

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11 Gb in size; the genome has a scaffold N50 value of 95.5 Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation.

SUMOylation facilitates the assembly of a Nuclear Factor-Y complex to enhance thermotolerance in Arabidopsis.

Heat stress (HS) has serious negative effects on plant development and has become a major threat to agriculture. A rapid transcriptional regulatory cascade has evolved in plants in response to HS. Nuclear Factor-Y (NF-Y) complexes are critical for this mechanism, but how NF-Y complexes are regulated remains unclear. In this study, we identified NF-YC10 (NF-Y subunit C10), a central regulator of the HS response in Arabidopsis thaliana, as a substrate of SUMOylation, an important post-translational modification. Biochemical analysis showed that the SUMO ligase SIZ1 (SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1) interacts with NF-YC10 and enhances its SUMOylation during HS. The SUMOylation of NF-YC10 facilitates its interaction with and the nuclear translocation of NF-YB3, in which the SUMO interaction motif (SIM) is essential for its efficient association with NF-YC10. Further functional analysis indicated that the SUMOylation of NF-YC10 and the SIM of NF-YB3 are critical for HS-responsive gene expression and plant thermotolerance. These findings uncover a role for the SIZ1-mediated SUMOylation of NF-YC10 in NF-Y complex assembly under HS, providing new insights into the role of a post-translational modification in regulating transcription during abiotic stress responses in plants.

Importation of chloroplast proteins under heat stress is facilitated by their SUMO conjugations.

Chloroplasts are hypersensitive to heat stress (HS). SUMOylation, a critical post-translational modification, is conservatively involved in HS responses. However, the functional connection between SUMOylation and chloroplasts under HS remains to be studied. The bioinformatics, biochemistry, and cell biology analyses were used to detect the SUMOylation statuses of Arabidopsis nuclear-encoded chloroplast proteins and the effect of SUMOylation on subcellular localization of these proteins under HS. PSBR, a subunit of photosystem II, was used as an example for a detailed investigation of functional mechanisms. After a global SUMOylation site prediction of nuclear-encoded chloroplast proteins, biochemical data showed that most of the selected candidates are modified by SUMO3 in the cytoplasm. The chloroplast localization of these SUMOylation targets under long-term HS is partially maintained by the SUMO ligase AtSIZ1. The HS-induced SUMOylation on PSBR contributes to the maintenance of its chloroplast localization, which is dependent on its chloroplast importation efficiency correlated to phosphorylation. The complementation analysis provided evidence that SUMOylation is essential for the physiological function of PSBR under HS. Our study illustrated a general regulatory mechanism of SUMOylation in maintaining the chloroplast protein importation during HS and provided hints for further investigation on protein modifications associated with plant organelles under stress conditions.

A SWI/SNF subunit regulates chromosomal dissociation of structural maintenance complex 5 during DNA repair in plant cells.

DNA damage decreases genome stability and alters genetic information in all organisms. Conserved protein complexes have been evolved for DNA repair in eukaryotes, such as the structural maintenance complex 5/6 (SMC5/6), a chromosomal ATPase involved in DNA double-strand break (DSB) repair. Several factors have been identified for recruitment of SMC5/6 to DSBs, but this complex is also associated with chromosomes under normal conditions; how SMC5/6 dissociates from its original location and moves to DSB sites is completely unknown. In this study, we determined that SWI3B, a subunit of the SWI/SNF complex, is an SMC5-interacting protein in Arabidopsis thialiana Knockdown of SWI3B or SMC5 results in increased DNA damage accumulation. During DNA damage, SWI3B expression is induced, but the SWI3B protein is not localized at DSBs. Notably, either knockdown or overexpression of SWI3B disrupts the DSB recruitment of SMC5 in response to DNA damage. Overexpression of a cotranscriptional activator ADA2b rescues the DSB localization of SMC5 dramatically in the SWI3B-overexpressing cells but only weakly in the SWI3B knockdown cells. Biochemical data confirmed that ADA2b attenuates the interaction between SWI3B and SMC5 and that SWI3B promotes the dissociation of SMC5 from chromosomes. In addition, overexpression of SMC5 reduces DNA damage accumulation in the SWI3B knockdown plants. Collectively, these results indicate that the presence of an appropriate level of SWI3B enhances dissociation of SMC5 from chromosomes for its further recruitment at DSBs during DNA damage in plant cells.

SUMOylation Stabilizes the Transcription Factor DREB2A to Improve Plant Thermotolerance.

Heat stress (HS) has serious effects on plant development, resulting in heavy agricultural losses. A critical transcription factor network is involved in plant adaptation to high temperature. DEHYDRATION RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) is a key transcription factor that functions in plant thermotolerance. The DREB2A protein is unstable under normal temperature and is degraded by the 26S proteasome; however, the mechanism by which DREB2A protein stability dramatically increases in response to HS remains poorly understood. In this study, we found that the DREB2A protein of Arabidopsis (Arabidopsis thaliana) is stabilized under high temperature by the posttranslational modification SUMOylation. Biochemical data indicated that DREB2A is SUMOylated at K163, a conserved residue adjacent to the negative regulatory domain during HS. SUMOylation of DREB2A suppresses its interaction with BPM2, a ubiquitin ligase component, consequently increasing DREB2A protein stability under high temperature. In addition, analysis of plant heat tolerance and marker gene expression indicated that DREB2A SUMOylation is essential for its function in the HS response. Collectively, our data reveal a role for SUMOylation in the maintenance of DREB2A stability under high temperature, thus improving our understanding of the regulatory mechanisms underlying HS response in plant cells.

Quantitative Fluorescence Resonance Energy Transfer Analysis on the Direct Interaction of Activation-2b with Histone H3/Switch-3B Protein in Arabidopsis Mesophyll Protoplasts.

Interaction between the alteration/deficiency in activation-2b (ADA2b) and histone H3/switch-3B (SWI3B) proteins was evaluated in arabidopsis mesophyll protoplasts by quantitative fluorescence resonance energy transfer (FRET) analysis. Microscopic image showed that ADA2b, SWI3B and H3 proteins colocalized in nucleus, and quantitative FRET measurements showed 0.31 of FRET efficiency (E) for the protoplasts coexpressing ECFP-ADA2b and EYFP-SWI3B, and 0.285 of E for the protoplasts coexpressing ECFP-H3 and EYFP-ADA2b, demonstrating the direct interaction of ADA2b with SWI3B/H3 protein. Collectively, SWI3B and H3 proteins are the inherent components of the ADA2b complex in which ADA2b directly interacts with SWI3B/H3 protein.

The Transcriptional Coactivator ADA2b Recruits a Structural Maintenance Protein to Double-Strand Breaks during DNA Repair in Plants.

DNA damage occurs in all cells and can hinder chromosome stability and cell viability. Structural Maintenance of Chromosomes5/6 (SMC5/6) is a protein complex that functions as an evolutionarily conserved chromosomal ATPase critical for repairing DNA double-strand breaks (DSBs). However, the mechanisms regulating this complex in plants are poorly understood. Here, we identified the transcriptional coactivator ALTERATION/DEFICIENCY IN ACTIVATION2B (ADA2b) as an interactor of SMC5 in Arabidopsis (Arabidopsis thaliana). ADA2b is a conserved component of the Spt-Ada-Gcn5 acetyltransferase complex, which functions in transcriptional regulation. Characterization of mutant and knockdown Arabidopsis lines showed that disruption of either SMC5 or ADA2b resulted in enhanced DNA damage. Both SMC5 and ADA2b were associated with γ-H2AX, a marker of DSBs, and the recruitment of SMC5 onto DSBs was dependent on ADA2b. In addition, overexpression of SMC5 in the ada2b mutant background stimulated cell death. Collectively, our results show that the interaction between ADA2b and SMC5 mediates DNA repair in plant cells, suggesting a functional association between these conserved proteins and further elucidating mechanisms of DNA damage repair in plants.

Post-translational modification: a strategic response to high temperature in plants.

With the increasing global warming, high-temperature stress is affecting plant growth and development with greater frequency. Therefore, an increasing number of studies examining the mechanism of temperature response contribute to a more optimal understanding of plant growth under environmental pressure. Post-translational modification (PTM) provides the rapid reconnection of transcriptional programs including transcription factors and signaling proteins. It is vital that plants quickly respond to changes in the environment in order to survive under stressful situations. Herein, we discuss several types of PTMs that occur in response to warm-temperature and high-temperature stress, including ubiquitination, SUMOylation, phosphorylation, histone methylation, and acetylation. This review provides a valuable resolution to this issue to enable increased crop productivity at high temperatures.

ABA INSENSITIVE 5 confers geminivirus resistance via suppression of the viral promoter activity in plants.

Geminiviruses are a large group of plant viruses that have been a serious threat to worldwide agriculture. Transcription of the virus-encoded genes is necessary for geminiviruses to complete their life cycle, but the host proteins which directly target geminivirus promoters for suppression of viral gene transcription remain to be identified. Using Beet severe curly top virus (BSCTV) which causes severe plant symptoms as a system, we performed a yeast one-hybrid screening and identified ABA INSENSITIVE 5 (ABI5), a critical transcription factor in Abscisic acid (ABA) signaling transduction, as an interactor with the viral promoter. Further data showed that an ABA-responsive element in the viral promoter is necessary for its interaction with ABI5 and symptom development. Overexpression of ABI5 suppresses the transcription activity of the viral promoter and BSCTV infection in Nicotiana benthamiana and Arabidopsis; whilst depletion of ABI5 enhances the infection of BSCTV in Arabidopsis. Taken together, our study uncovered the function of ABI5 in the plant-virus interaction and will provide us with a new strategy to protect crops from geminivirus infection.

SUMOylation: A critical transcription modulator in plant cells.

Gene transcription is critical for various cellular processes and is precisely controlled at multiple levels, and posttranslational modification (PTM) is a fast and powerful way to regulate transcription factors (TFs). SUMOylation, which conjugates small ubiquitin-related modifier (SUMO) molecules to protein substrates, is a crucial PTM that modulates the activity, stability, subcellular localization, and partner interactions of TFs in plant cells. Here, we summarize the mechanisms of SUMOylation in the regulation of transcription in plant development and stress responses. We also discuss the crosstalk between SUMOylation and other PTMs, as well as the potential functions of SUMOylation in the regulation of transcription-associated complexes on plant chromatin. This summary and perspective will improve understanding of the molecular mechanism of PTMs in plant transcription regulation.

Chromatin-associated SUMOylation controls the transcriptional switch between plant development and heat stress responses.

The post-translational protein modification known as SUMOylation has conserved roles in the heat stress responses of various species. The functional connection between the global regulation of gene expression and chromatin-associated SUMOylation in plant cells is unknown. Here, we uncovered a genome-wide relationship between chromatin-associated SUMOylation and transcriptional switches in Arabidopsis thaliana grown at room temperature, exposed to heat stress, and exposed to heat stress followed by recovery. The small ubiquitin-like modifier (SUMO)-associated chromatin sites, characterized by whole-genome ChIP-seq, were generally associated with active chromatin markers. In response to heat stress, chromatin-associated SUMO signals increased at promoter-transcriptional start site regions and decreased in gene bodies. RNA-seq analysis supported the role of chromatin-associated SUMOylation in transcriptional activation during rapid responses to high temperature. Changes in SUMO signals on chromatin were associated with the upregulation of heat-responsive genes and the downregulation of growth-related genes. Disruption of the SUMO ligase gene SIZ1 abolished SUMO signals on chromatin and attenuated rapid transcriptional responses to heat stress. The SUMO signal peaks were enriched in DNA elements recognized by distinct groups of transcription factors under different temperature conditions. These observations provide evidence that chromatin-associated SUMOylation regulates the transcriptional switch between development and heat stress response in plant cells.

AtMMS21: Connecting DNA Repair and Root Development.

Two recent reports show that SUMO ligase AtMMS21 controls the cell cycle through dissociating the E2Fa/DPa complex, and regulates chromatin remodeling by maintaining the stability of BRAHMA. We discuss these novel functions of AtMMS21 and its potential role in linking DNA repair and root development.

The SWI/SNF subunit SWI3B regulates IAMT1 expression via chromatin remodeling in Arabidopsis leaf development.

The SWI/SNF complex is crucial to chromatin remodeling in various biological processes in different species, but the distinct functions of its components in plant development remain unclear. Here we uncovered the role of SWI3B, a subunit of the Arabidopsis thaliana SWI/SNF complex, via RNA interference. Knockdown of SWI3B resulted in an upward-curling leaf phenotype. Further investigation showed that the RNA level of IAA carboxyl methyltransferase 1 (IAMT1), encoding an enzyme involved in auxin metabolism, was dramatically elevated in the knockdown (SWI3B-RNAi) plants. In addition, activation of IAMT1 produced a leaf-curling phenotype similar to that of the SWI3B-RNAi lines. Database analysis suggested that the last intron of IAMT contains a site of polymerase V (Pol V) stabilized nucleosome, which may be associated with SWI3B. Data from a micrococcal nuclease (MNase) digestion assay showed that nucleosome occupancy around this site was downregulated in the leaves of SWI3B-RNAi plants. In addition, knockdown of IAMT1 in the SWI3B-RNAi background repressed the abnormal leaf development. Thus, SWI3B-mediated chromatin remodeling is critical in regulating the expression of IAMT1 in leaf development.