Soybean AROGENATE DEHYDRATASES (GmADTs): involvement in the cytosolic isoflavonoid metabolon or trans-organelle continuity?

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon.

MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa.

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa's relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9-overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate.

Plant production of a virus-like particle-based vaccine candidate against porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome (PRRS) is a disease leading to spontaneous abortions and stillbirths in sows and lowered life quality and expectancy in growing pigs. PRRS is prevalent worldwide and has significant economic impacts to swine industries around the globe. Co-expression of the two most abundant proteins in the viral envelope, the matrix protein (M) and glycosylated protein 5 (GP5), can produce a neutralizing immune response for the virus providing a potentially effective subunit vaccine against the disease, but these proteins are difficult to express. The goal of this research was to display antigenic portions of the M and GP5 proteins on the surface of tobacco mosaic virus-like particles. A modified tobacco mosaic virus coat protein (TMVc) was transiently expressed in Nicotiana benthamiana leaves and targeted to three subcellular compartments along the secretory pathway to introduce glycosylation patterns important for M-GP5 epitope immunogenicity. We found that accumulation levels in the apoplast were similar to the ER and the vacuole. Because glycans present on plant apoplastic proteins are closest to those present on PRRSV proteins, a TMVc-M-GP5 fusion construct was targeted to the apoplast and accumulated at over 0.5 mg/g of plant fresh weight. TMVc virus-like particles self-assembled in plant cells and surface-displayed the M-GP5 epitope, as visualized by transmission electron microscopy and immunogold localization. These promising findings lay the foundation for immunogenicity and protective-immunity studies in animals to examine the efficacy of this vaccine candidate as a measure to control PRRS.

Localization of Arabidopsis Glucan Synthase-Like 5, 8, and 12 to plasmodesmata and the GSL8-dependent role of PDLP5 in regulating plasmodesmal permeability.

Cell-to-cell communication via membranous channels called plasmodesmata (PD) plays critical roles during plant development and in response to biotic and abiotic stresses. Several enzymes and receptor-like proteins (RLPs), including Arabidopsis thaliana glucan synthase-likes (GSLs), also known as callose synthases (CALSs), and PD-located proteins (PDLPs), have been implicated in plasmodesmal permeability regulation and intercellular communication. Localization of PDLPs to punctate structures at the cell periphery and their receptor-like identity have raised the hypothesis that PDLPs are involved in the regulation of symplastic trafficking during plant development and in response to endogenous and exogenous signals. Indeed, it was shown that PDLP5 could limit plasmodesmal permeability through inducing an increase in callose accumulation at PD. However, mechanistically, how this is achieved remains to be elucidated. To address this key issue in understanding the regulation of PD, physical and functional interactions between PDLPs and GSLs (using the PDLP5-GSL8/CALS10 pair as a model) were investigated. Our results show that GSL8/CALS10 plays essential roles and is required for the function and plasmodesmal localization of PDLP5. Furthermore, it was demonstrated that the localization of PDLP5 to PD and its function in inducing callose deposition are GSL8-dependent. Importantly, our transgenic study shows that three key members of the GSL family, i.e., GSL5/CALS12, GSL8/CALS10, and GSL12/CALS3, localize to PD and co-localize with PDLP5, suggesting that GSL8/CALS10 might not be the only callose synthase with the determining role in PD regulation. These findings, together with our previous observation showing the direct interaction of GSL8/CALS10 with PDLP5, indicate the pivotal role of the GSL8/CALS10-PDLP5 interplay in regulating PD permeability. Future work is needed to investigate whether the PDLP5 functionality and localization are also disrupted in gsl5 and gsl12, or it is just gsl8-specific.

SPL12 Regulates AGL6 and AGL21 to Modulate Nodulation and Root Regeneration under Osmotic Stress and Nitrate Sufficiency Conditions in Medicago sativa.

The highly conserved plant microRNA, miR156, affects root architecture, nodulation, symbiotic nitrogen fixation, and stress response. In Medicago sativa, transcripts of eleven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE, SPLs, including SPL12, are targeted for cleavage by miR156. Our previous research revealed the role of SPL12 and its target gene, AGL6, in nodulation in alfalfa. Here, we investigated the involvement of SPL12, AGL6 and AGL21 in nodulation under osmotic stress and different nitrate availability conditions. Characterization of phenotypic and molecular parameters revealed that the SPL12/AGL6 module plays a negative role in maintaining nodulation under osmotic stress. While there was a decrease in the nodule numbers in WT plants under osmotic stress, the SPL12-RNAi and AGL6-RNAi genotypes maintained nodulation under osmotic stress. Moreover, the results showed that SPL12 regulates nodulation under a high concentration of nitrate by silencing AGL21. AGL21 transcript levels were increased under nitrate treatment in WT plants, but SPL12 was not affected throughout the treatment period. Given that AGL21 was significantly upregulated in SPL12-RNAi plants, we conclude that SPL12 may be involved in regulating nitrate inhibition of nodulation in alfalfa by targeting AGL21. Taken together, our results suggest that SPL12, AGL6, and AGL21 form a genetic module that regulates nodulation in alfalfa under osmotic stress and in response to nitrate.

Deciphering the role of SPL12 and AGL6 from a genetic module that functions in nodulation and root regeneration in Medicago sativa.

KEY MESSAGE: Our results show that SPL12 plays a crucial role in regulating nodule development in Medicago sativa L. (alfalfa), and that AGL6 is targeted and downregulated by SPL12. Root architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and response to biotic and abiotic stress factors. The small RNA, microRNA156 (miR156), is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing miR156 display increased nodulation, improved nitrogen fixation and enhanced root regenerative capacity during vegetative propagation. In alfalfa, transcripts of eleven SPLs, including SPL12, are targeted for cleavage by miR156. In this study, we characterized the role of SPL12 in root architecture and nodulation by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and -overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 which encodes AGAMOUS-like MADS box transcription factor, was identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Taken together, our results suggest that SPL12 and AGL6 form a genetic module that regulates root development and nodulation in alfalfa.

Purification and Proteomics Analysis of Phloem Tissues from Virus-Infected Plants.

The plant phloem vasculature is crucial for plant growth and development, and is essential for the systemic movement (SM) of plant viruses. Recent transcriptomic studies of the phloem during virus infection have shown the importance of this tissue, yet transcript levels do not provide definitive answers how virus-host interactions favour successful viral SM. Proteomic analyses have been used to identify host-virus protein interactions, uncovering a variety of ways by which viruses utilize host cellular machinery for completion of the viral infection cycle. Despite this new evidence through proteomics, very few phloem centric studies during viral infection have been performed. Here, we describe a protocol for the isolation of phloem tissues and proteins and the subsequent label-free quantitation (LFQ), for identification of proteomic alterations caused by viral infection.

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone.

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

LEAFY COTYLEDON1 expression in the endosperm enables embryo maturation in Arabidopsis.

The endosperm provides nutrients and growth regulators to the embryo during seed development. LEAFY COTYLEDON1 (LEC1) has long been known to be essential for embryo maturation. LEC1 is expressed in both the embryo and the endosperm; however, the functional relevance of the endosperm-expressed LEC1 for seed development is unclear. Here, we provide genetic and transgenic evidence demonstrating that endosperm-expressed LEC1 is necessary and sufficient for embryo maturation. We show that endosperm-synthesized LEC1 is capable of orchestrating full seed maturation in the absence of embryo-expressed LEC1. Inversely, without LEC1 expression in the endosperm, embryo development arrests even in the presence of functional LEC1 alleles in the embryo. We further reveal that LEC1 expression in the endosperm begins at the zygote stage and the LEC1 protein is then trafficked to the embryo to activate processes of seed maturation. Our findings thus establish a key role for endosperm in regulating embryo development.

Involvement of the miR156/SPL module in flooding response in Medicago sativa.

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

Genome-wide occupancy of Arabidopsis SWI/SNF chromatin remodeler SPLAYED provides insights into its interplay with its close homolog BRAHMA and Polycomb proteins.

SPLAYED (SYD) is a SWItch/Sucrose Non-Fermentable (SWI/SNF)-type chromatin remodeler identified in Arabidopsis thaliana (Arabidopsis). It is believed to play both redundant and differential roles with its closest homolog BRAHMA (BRM) in diverse plant growth and development processes. To better understand how SYD functions, we profiled the genome-wide occupancy of SYD and its impact on the global transcriptome and trimethylation of histone H3 on lysine 27 (H3K27me3). To map the global occupancy of SYD, we generated a GFP-tagged transgenic line and used it for chromatin immunoprecipitation experiments followed by next-generation sequencing, by which more than 6000 SYD target genes were identified. Through integrating SYD occupancy and transcriptome profiles, we found that SYD preferentially targets to nucleosome-free regions of expressed genes. Further analysis revealed that SYD occupancy peaks exhibit five distinct patterns, which were also shared by BRM and BAF60, a conserved SWI/SNF complex component, indicating the common target sites of these SWI/SNF chromatin remodelers and the functional relevance of such distinct patterns. To investigate the interplay between SYD and Polycomb-group (PcG) proteins, we performed a genome-wide analysis of H3K27me3 in syd-5. We observed both increases and decreases in H3K27me3 levels at a few hundred genes in syd-5 compared to wild type. Our results imply that SYD can act antagonistically or synergistically with PcG at specific genes. Together, our SYD genome-wide occupancy data and the transcriptome and H3K27me3 profiles provide a much-needed resource for dissecting SYD's crucial roles in the regulation of plant growth and development.

Transcriptome-IPMS analysis reveals a tissue-dependent miR156/SPL13 regulatory mechanism in alfalfa drought tolerance.

BACKGROUND: We previously reported on the interplay between miR156/SPL13 and WD40-1/DFR to improve response to drought stress in alfalfa (Medicago sativa L.). Here we aimed to investigate whether the role of miR156/SPL13 module in drought response is tissue-specific, and to identify SPL13-interacting proteins. We analyzed the global transcript profiles of leaf, stem, and root tissues of one-month old RNAi-silenced SPL13 (SPL13RNAi) alfalfa plants exposed to drought stress and conducted protein-protein interaction analysis to identify SPL13 interacting partners. RESULT: Transcript analysis combined with weighted gene co-expression network analysis showed tissue and genotype-specific gene expression patterns. Moreover, pathway analysis of stem-derived differentially expressed genes (DEG) revealed upregulation of genes associated with stress mitigating primary and specialized metabolites, whereas genes associated with photosynthesis light reactions were silenced in SPL13RNAi plants. Leaf-derived DEG were attributed to enhanced light reactions, largely photosystem I, II, and electron transport chains, while roots of SPL13RNAi plants upregulated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. Using immunoprecipitation combined with mass spectrometry (IPMS) we showed that SPL13 interacts with proteins involved in photosynthesis, specialized metabolite biosynthesis, and stress tolerance. CONCLUSIONS: We conclude that the miR156/SPL13 module mitigates drought stress in alfalfa by regulating molecular and physiological processes in a tissue-dependent manner.

Evidence that AGL17 is a significant downstream target of CLF in floral transition control.

Polycomb-group (PcG) proteins are evolutionarily conserved in higher organisms and play essential roles in many developmental processes by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3), a key repressive histone mark. In Arabidopsis (Arabidopsis thaliana), histone methyltransferase CURLY LEAF (CLF) is one of the major PcG catalytic components, playing critical roles in plant growth and development, especially the floral transition. We have recently profiled the genome-wide occupancy of CLF by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Interestingly, AGAMOUS-LIKE 17 (AGL17) that encodes a known flowering activator was found to be a CLF direct target. Based on this observation, we set out to examine to what extent this genetic module regulates the flowering. First, we validated the ChIP-seq results by ChIP-qPCR and show that CLF indeed targets AGL17, and the level of H3K27me3 is decreased when CLF is lost. Further, we show that the expression of AGL17 is significantly up-regulated in the clf-29 mutant compared to wild-type (WT). Finally, our clf agl17 double mutant analysis suggests that AGL17 is a significant downstream target of CLF in floral transition control.

The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa.

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Genome-wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome-wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP-tagged CLF/SWN under their respective native promoters and used them for ChIP-seq analyses to profile the genome-wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild-type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA-like and Telo-box-like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis.

BACKGROUND: Plant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood. RESULTS: CALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a ß-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata. CONCLUSION: Analysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation.

Moonlighting proteins: putting the spotlight on enzymes.

AROGENATE DEHAYDRATASE2 (ADT2) is a member of the Arabidopsis thaliana ADT family. All members of this family act as arogenate dehydratases in phenylalanine biosynthesis, decarboxylating/dehydrating arogenate to phenylalanine. ADT2 is detected in stromules, and as a ring around the equatorial plane of dividing chloroplasts, indicating it has a second, non-enzymatic function in chloroplast division. Here, we provide further evidence for this alternative role of ADT2. First, we demonstrate that ADT2 and FtsZ co-localize around the equatorial plane at the same time. Second, FtsZ expression in an adt2 mutant was analyzed, as well as ADT2 expression in three Arabidopsis chloroplast division mutants, ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), ARC5 and ARC6. In arc3 and arc6 mutants, ADT2 is misexpressed and resembles the expression of FtsZ in the same mutants. However, in the arc5 mutant, ADT2 ring positioning is observed at constriction points indicating proper relative timing. ADT2 expression in the arc mutants shows that the role of ADT2 in chloroplast division occurs prior to ARC5, but is dependent on ARC3 and ARC6. Abbreviations used: ADT: arogenate dehydratase, ARC: accumulation and replication of chloroplasts, CFP: cyan fluorescent protein, dpi: days post infiltration, FtsZ: filamentous temperature sensitive Z, PD: plastid division, Phe: phenylalanine, YFP: yellow fluorescent protein.

Cytosolic acetyl-CoA promotes histone acetylation predominantly at H3K27 in Arabidopsis.

Acetyl-coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation, particularly histone acetylation that promotes gene expression. However, the effect of acetyl-CoA levels on histone acetylation status in plants remains unknown. Here, we show that malfunctioned cytosolic acetyl-CoA carboxylase1 (ACC1) in Arabidopsis leads to elevated levels of acetyl-CoA and promotes histone hyperacetylation predominantly at lysine 27 of histone H3 (H3K27). The increase of H3K27 acetylation (H3K27ac) is dependent on adenosine triphosphate (ATP)-citrate lyase which cleaves citrate to acetyl-CoA in the cytoplasm, and requires histone acetyltransferase GCN5. A comprehensive analysis of the transcriptome and metabolome in combination with the genome-wide H3K27ac profiles of acc1 mutants demonstrate the dynamic changes in H3K27ac, gene transcripts and metabolites occurring in the cell by the increased levels of acetyl-CoA. This study suggests that H3K27ac is an important link between cytosolic acetyl-CoA level and gene expression in response to the dynamic metabolic environments in plants.