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Flooding impairs plant growth through oxygen deprivation, which activates plant survival and acclimation responses. Transcriptional responses to low oxygen are generally associated with the activation of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors. However, the exact mechanisms and molecular components by which ERFVII factors initiate gene expression are not fully elucidated. Here, we show that the ERFVII factors RELATED TO APETALA 2.2 (RAP2.2) and RAP2.12 cooperate with the Mediator complex subunit AtMED25 to coordinate gene expression under hypoxia in Arabidopsis thaliana. Respective med25 knock-out mutants display reduced low-oxygen stress tolerance. AtMED25 physically associates with a distinct set of hypoxia core genes and its loss partially impairs transcription under hypoxia due to decreased RNA polymerase II recruitment. Association of AtMED25 with target genes requires the presence of ERFVII transcription factors. Next to ERFVII protein stabilisation, also the composition of the Mediator complex including AtMED25 is potentially affected by hypoxia stress as shown by protein-complex pulldown assays. The dynamic response of the Mediator complex to hypoxia is furthermore supported by the fact that two subunits, AtMED8 and AtMED16, are not involved in the establishment of hypoxia tolerance, whilst both act in coordination with AtMED25 under other environmental conditions. We furthermore show that AtMED25 function under hypoxia is independent of ethylene signalling. Finally, functional conservation at the molecular level was found for the MED25-ERFVII module between A. thaliana and the monocot species Oryza sativa, pointing to a potentially universal role of MED25 in coordinating ERFVII-dependent transcript responses to hypoxia in plants., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants have evolved and adapted under dynamic environmental conditions, particularly to fluctuating light, but plant research has often focused on constant growth conditions. To quantitatively asses the adaptation to fluctuating light, a panel of 384 natural Arabidopsis thaliana accessions was analyzed in two parallel independent experiments under fluctuating and constant light conditions in an automated high-throughput phenotyping system upgraded with supplemental LEDs. While the integrated daily photosynthetically active radiation was the same under both light regimes, plants in fluctuating light conditions accumulated significantly less biomass and had lower leaf area during their measured vegetative growth than plants in constant light. A total of 282 image-derived architectural and/or color-related traits at six common time points, and 77 photosynthesis-related traits from one common time point were used to assess their associations with genome-wide natural variation for both light regimes. Out of the 3000 significant marker-trait associations (MTAs) detected, only 183 (6.1%) were common for fluctuating and constant light conditions. The prevalence of light regime-specific QTL indicates a complex adaptation. Genes in linkage disequilibrium with fluctuating light-specific MTAs with an adjusted repeatability value >0.5 were filtered for gene ontology terms containing "photo" or "light", yielding 15 selected candidates. The candidate genes are involved in photoprotection, PSII maintenance and repair, maintenance of linear electron flow, photorespiration, phytochrome signaling, and cell wall expansion, providing a promising starting point for further investigations into the response of Arabidopsis thaliana to fluctuating light conditions., (© 2024 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Many non-graminaceous species release various coumarins in response to iron (Fe) deficiency. However, the physiological relevance of these coumarins remains poorly understood. Here, we show that the three enzymes leading to sideretin biosynthesis co-exist in Arabidopsis (Arabidopsis thaliana) epidermal and cortical cells and that the shift to fraxetin at alkaline pH depends on MYB72-mediated repression of CYTOCHROME P450, FAMILY 82, SUBFAMILY C, POLYPEPTIDE 4 (CYP82C4). In vitro, only fraxetin and sideretin can reduce part of the Fe(III) that they mobilize. We demonstrate that coumarin-mediated Fe(III) reduction is critical under acidic conditions, as fraxetin and sideretin can complement the Fe(III)-chelate reductase mutant ferric reduction oxidase 2 (fro2), and disruption of coumarin biosynthesis in fro2 plants impairs Fe acquisition similar to in the Fe(II) uptake-deficient mutant iron-regulated transporter 1 (irt1). Disruption of sideretin biosynthesis in a fro2 cyp82C4-1 double mutant revealed that sideretin is the dominant chemical reductant that functions with FRO2 to mediate Fe(II) formation for root uptake. At alkaline pH, Fe(III) reduction by coumarins becomes almost negligible but fraxetin still sustains high Fe(III) mobilization, suggesting that its main function is to provide chelated Fe(III) for FRO2. Our study indicates that strategy-I plants link sideretin and fraxetin biosynthesis and secretion to external pH to recruit distinct coumarin chemical activities to maximize Fe acquisition according to prevailing soil pH conditions., Competing Interests: Conflict of interest statement. The authors declare no conflict of interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Temperature-induced elongation of hypocotyls, petioles, and roots, together with hyponastic leaf responses, constitute key model phenotypes that can be used to assess a plant's capacity for thermomorphogenesis. Phenotypic responses are often quantified at a single time point during seedling development at different temperatures. However, to capture growth dynamics, several time points need to be assessed, and ideally continuous measurements are taken. Here we describe a general experimental setup and technical solutions for recording and measuring seedling phenotypes at single and multiple time points. Furthermore, we present an R-package called "rootdetectR," which allows easy processing of hypocotyl, root or petiole length, and growth rate data and provides different options of data presentation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

As a major determinant of the nutrient-acquiring root surface, root hairs (RHs) provide a low-input strategy to enhance nutrient uptake. Although primary and lateral roots exhibit elongation responses under mild nitrogen (N) deficiency, the foraging response of RHs and underlying regulatory mechanisms remain elusive. Employing transcriptomics and functional studies revealed a framework of molecular components composing a cascade of auxin synthesis, transport, and signaling that triggers RH elongation for N acquisition. Through upregulation of Tryptophan Aminotransferase of Arabidopsis 1 (TAA1) and YUCCA8, low N increases auxin accumulation in the root apex. Auxin is then directed to the RH differentiation zone via the auxin transport machinery, AUXIN TRANSPORTER PROTEIN 1 (AUX1) and PIN-FORMED 2 (PIN2). Upon arrival to the RH zone, auxin activates the transcription factors AUXIN RESPONSE FACTOR 6 and 8 (ARF6/8) to promote the epidermal and auxin-inducible transcriptional module ROOT HAIR DEFECTIVE 6 (RHD6)-LOTUS JAPONICA ROOT HAIRLESS-LIKE 3 (LRL3) to steer RH elongation in response to low N. Our study uncovers a spatially defined regulatory signaling cascade for N foraging by RHs, expanding the mechanistic framework of hormone-regulated nutrient sensing in plant roots., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Background: Plant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens., Results: Here, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000., Conclusion: We demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses., (© 2023. The Author(s).)

During meiotic prophase I, sister chromatids are arranged in a loop-base array along a proteinaceous structure, called the meiotic chromosome axis. This structure is essential for synapsis and meiotic recombination progression and hence formation of genetically diverse gametes. Proteomic studies in plants aiming to unravel the composition and regulation of meiotic axes are constrained by limited meiotic cells embedded in floral organs. Here we report TurboID (TbID)-based proximity labelling (PL) in meiotic cells of Arabidopsis thaliana. TbID fusion to the two meiotic chromosome axis proteins ASY1 and ASY3 enabled the identification of their proximate 'interactomes' based on affinity purification coupled with mass spectrometry. We identified 39 ASY1 and/or ASY3 proximate candidates covering most known chromosome axis-related proteins. Functional studies of selected candidates demonstrate that not only known meiotic candidates but also new meiotic proteins were uncovered. Hence, TbID-based PL in meiotic cells enables the identification of chromosome axis proximate proteins in A. thaliana., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

Fluorescence live-cell microscopy is important in cell biology to perform artifact-free investigations. To analyze the dynamics of chromatin and centromeres at different stages of the cell cycle in nuclei and chromosomes, we performed simultaneous EYFP-CENH3/H2B-DsRed and single H2B-YFP transformations in Arabidopsis wild-type and cohesin T-DNA mutants. All constructs were under the control of the strong CaMV 35S promoter. While a strong silencing of fluorescence expression occurred differently in leaf and root tissues in the double transformants, nearly all single-transformed wild-type and most mutant cells showed H2B-YFP fluorescence. It seems that for an efficient co-expression of two fluorescence proteins, endogenous promoters and terminators should be used., (© 2023 S. Karger AG, Basel.)

Despite serving as a major inorganic nitrogen source for plants, ammonium causes toxicity at elevated concentrations, inhibiting root elongation early on. While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species (ROS) in roots, it remains unclear about the mechanisms underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium. In this study, we demonstrate that ammonium-induced apoplastic acidification co-localizes with Fe precipitation and hydrogen peroxide (H 2 O 2 ) accumulation along the stele of the elongation and differentiation zone in root tips, indicating Fe-dependent ROS formation. By screening ammonium sensitivity in T-DNA insertion lines of ammonium-responsive genes, we identified PDX1.1, which is upregulated by ammonium in the root stele and whose product catalyzes de novo biosynthesis of vitamin B 6 . Root growth of pdx1.1 mutants is hypersensitive to ammonium, while chemical complementation or overexpression of PDX1.1 restores root elongation. This salvage strategy requires non-phosphorylated forms of vitamin B 6 that are able to quench ROS and rescue root growth from ammonium inhibition. Collectively, these results suggest that PDX1.1-mediated synthesis of non-phosphorylated B 6 vitamers acts as a primary strategy to protect roots from ammonium-dependent ROS formation., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

In plants, the antagonism between growth and defense is hardwired by hormonal signaling. The perception of pathogen-associated molecular patterns (PAMPs) from invading microorganisms inhibits auxin signaling and plant growth. Conversely, pathogens manipulate auxin signaling to promote disease, but how this hormone inhibits immunity is not fully understood. Ustilago maydis is a maize pathogen that induces auxin signaling in its host. We characterized a U. maydis effector protein, Naked1 (Nkd1), that is translocated into the host nucleus. Through its native ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, Nkd1 binds to the transcriptional co-repressors TOPLESS/TOPLESS-related (TPL/TPRs) and prevents the recruitment of a transcriptional repressor involved in hormonal signaling, leading to the de-repression of auxin and jasmonate signaling and thereby promoting susceptibility to (hemi)biotrophic pathogens. A moderate upregulation of auxin signaling inhibits the PAMP-triggered reactive oxygen species (ROS) burst, an early defense response. Thus, our findings establish a clear mechanism for auxin-induced pathogen susceptibility. Engineered Nkd1 variants with increased expression or increased EAR-mediated TPL/TPR binding trigger typical salicylic-acid-mediated defense reactions, leading to pathogen resistance. This implies that moderate binding of Nkd1 to TPL is a result of a balancing evolutionary selection process to enable TPL manipulation while avoiding host recognition., (© 2021 The Authors.)

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL , regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM , two ESR genes, and one of the HD-ZIP III members, REV , whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial-abaxial polarity is observed in wus   esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves.

Storage of meristematic tissue at ultra-low temperatures offers a mean to maintain valuable genetic resources from vegetatively reproduced plants. To reveal the biology underlying cryo-stress, shoot tips of the model plant Arabidopsis thaliana were subjected to a standard preservation procedure. A transcriptomic approach was taken to describe the subsequent cellular events which occurred. The cryoprotectant treatment induced the changes in the transcript levels of genes associated with RNA processing and primary metabolism. Explants of a mutant lacking a functional copy of the transcription factor WRKY22 were compromised for recovery. A number of putative downstream targets of WRKY22 were identified, some related to phytohormone-mediated defense, to the osmotic stress response, and to development. There were also alterations in the abundance of transcript produced by genes encoding photosynthesis-related proteins. The wrky22 mutant plants developed an open stomata phenotype in response to their exposure to the cryoprotectant solution. WRKY22 probably regulates a transcriptional network during cryo-stress, linking the explant's defense and osmotic stress responses to changes in its primary metabolism. A model is proposed linking WRKY53 and WRKY70 downstream of the action of WRKY22., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background and Aims: The stomatal conductance (gs) of most plant species decreases in response to elevated atmospheric CO2 concentration. This response could have a significant impact on plant water use in a future climate. However, the regulation of the CO2-induced stomatal closure response is not fully understood. Moreover, the potential genetic links between short-term (within minutes to hours) and long-term (within weeks to months) responses of gs to increased atmospheric CO2 have not been explored., Methods: We used Arabidopsis thaliana recombinant inbred lines originating from accessions Col-0 (strong CO2 response) and C24 (weak CO2 response) to study short- and long-term controls of gs. Quantitative trait locus (QTL) mapping was used to identify loci controlling short- and long-term gs responses to elevated CO2, as well as other stomata-related traits., Key Results: Short- and long-term stomatal responses to elevated CO2 were significantly correlated. Both short- and long-term responses were associated with a QTL at the end of chromosome 2. The location of this QTL was confirmed using near-isogenic lines and it was fine-mapped to a 410-kb region. The QTL did not correspond to any known gene involved in stomatal closure and had no effect on the responsiveness to abscisic acid. Additionally, we identified numerous other loci associated with stomatal regulation., Conclusions: We identified and confirmed the effect of a strong QTL corresponding to a yet unknown regulator of stomatal closure in response to elevated CO2 concentration. The correlation between short- and long-term stomatal CO2 responses and the genetic link between these traits highlight the importance of understanding guard cell CO2 signalling to predict and manipulate plant water use in a world with increasing atmospheric CO2 concentration. This study demonstrates the power of using natural variation to unravel the genetic regulation of complex traits., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company.)

In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.

TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.

Gramineous plants take up silicon (Si) that enhances the formation of exodermal Casparian bands (CBs) in the roots of rice (Oryza sativa L.). Furthermore, it is known that Si supply reduces the concentration of Fe in rice shoots. We hypothesized that the Si-enhanced CB formation in the exodermis reduces in the flux of Fe in the apoplast and the uptake of Fe loaded deoxymugineic acid. Thus, the effect of silicic acid supply at varied Fe concentrations and Fe forms was investigated in nutrient solution. The Fe concentrations in the shoot and apoplastic Fe concentrations in the root were determined and an Affymetrix GeneChip experiment was carried out together with qRT-PCR measurements for observation of transcriptomic reactions. Additionally, the Fe uptake of an overexpression mutant of OsABCG25 with an enhanced exodermal CB formation was investigated. The application of silicic acid reduced the Fe concentrations in shoot DM independently of the supplied Fe concentration and Fe form. As a reaction to the Fe shortage, the full cascade of Fe-homeostasis-related genes in the roots was upregulated. Silicic acid supply also decreased the apoplastic Fe concentrations in roots. In addition, an overexpression mutant of OsABCG25 with an enhanced CB formation showed a reduced uptake of Fe in excess Fe conditions. The results suggest that the Si-induced CB formation in the exodermis hampers the flux of Fe into the apoplast of the cortex and, thus, Fe uptake of rice grown in nutrient solution which is reflected in the upregulation of Fe homeostasis-related genes.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASP SIM3 is co-expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N-terminal tail and the histone fold domain of non-nucleosomal CenH3. Reduced NASP SIM3 expression negatively affects CenH3 deposition, identifying NASP SIM3 as a CenH3 histone chaperone., (© 2019 Leibniz Institute of Plant genetics and Crop Plant Research (IPK). The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.

Plants continuously need to adapt to their environment and prioritize either growth or defense responses to secure survival and reproduction. Trade-offs between growth and defense are often attributed to the allocation of energy for growth to adaptation responses. Still, the exact mechanisms underlying growth and defense trade-offs are poorly understood. Here, we demonstrate that the growth-related transcription factor HOMOLOG OF BEE2 INTERACTING WITH IBH 1 (HBI1) regulates apoplastic reactive oxygen species (ROS) homeostasis by differentially controlling the expression of NADPH oxidases (NOXs) and peroxidases (POXs). The HBI1 target genes RESPIRATORY BURST OXIDASE HOMOLOG A (RbohA) and RbohC have contrasting effects on the regulation of cell size. In addition, the HBI1-controlled NOXs and POXs oppositely regulate susceptibility toward Pseudomonas syringae. Our findings reveal that the incompatibility between growth and defense programs can be attributed to the way apoplastic ROS homeostasis is modulated during both processes., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Developmental plasticity of root system architecture is crucial for plant performance in nutrient-poor soils. Roots of plants grown under mild nitrogen (N) deficiency show a foraging response characterized by increased root length but mechanisms underlying this developmental plasticity are still elusive. By employing natural variation in Arabidopsis accessions, we show that the brassinosteroid (BR) signaling kinase BSK3 modulates root elongation under mild N deficiency. In particular, a proline to leucine substitution in the predicted kinase domain of BSK3 enhances BR sensitivity and signaling to increase the extent of root elongation. We further show that low N specifically upregulates transcript levels of the BR co-receptor BAK1 to activate BR signaling and stimulate root elongation. Altogether, our results uncover a role of BR signaling in root elongation under low N. The BSK3 alleles identified here provide targets for improving root growth of crops growing under limited N conditions.

Plant cytokinesis involves membrane trafficking and cytoskeletal rearrangements. Here, we report that the phosphoinositide kinases PI4Kβ1 and PI4Kβ2 integrate these processes in Arabidopsis thaliana ( Arabidopsis ) roots. Cytokinetic defects of an Arabidopsis pi4kβ1 pi4kβ2 double mutant are accompanied by defects in membrane trafficking. Specifically, we show that trafficking of the proteins KNOLLE and PIN2 at the cell plate, clathrin recruitment, and endocytosis is impaired in pi4kβ1 pi4kβ2 double mutants, accompanied by unfused vesicles at the nascent cell plate and around cell wall stubs. Interestingly, pi4kβ1 pi4kβ2 plants also display ectopic overstabilization of phragmoplast microtubules, which guide membrane trafficking at the cell plate. The overstabilization of phragmoplasts in the double mutant coincides with mislocalization of the microtubule-associated protein 65-3 (MAP65-3), which cross-links microtubules and is a downstream target for inhibition by the MAP kinase MPK4. Based on similar cytokinetic defects of the pi4kβ1 pi4kβ2 and mpk4-2 mutants and genetic and physical interaction of PI4Kβ1 and MPK4, we propose that PI4Kβ and MPK4 influence localization and activity of MAP65-3, respectively, acting synergistically to control phragmoplast dynamics., (© 2019 The Authors.)

Plant-specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY-YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators. Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana. Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET-specific differentially expressed genes and mostly independently characteristic, ET-specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects. The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods., (© 2018 John Wiley & Sons Ltd.)

Chlorophyll is synthesized from activated glutamate in the tetrapyrrole biosynthesis pathway through at least 20 different enzymatic reactions. Among these, the MgProto monomethylester (MgProtoME) cyclase catalyzes the formation of a fifth isocyclic ring to tetrapyrroles to form protochlorophyllide. The enzyme consists of two proteins. The CHL27 protein is proposed to be the catalytic component, while LCAA/YCF54 likely acts as a scaffolding factor. In comparison to other reactions of chlorophyll biosynthesis, this enzymatic step lacks clear elucidation and it is hardly understood, how electrons are delivered for the NADPH-dependent cyclization reaction. The present study intends to elucidate more precisely the role of LCAA/YCF54. Transgenic Arabidopsis lines with inactivated and overexpressed YCF54 reveal the mutual stability of YCF54 and CHL27. Among the YCF54-interacting proteins, the plastidal ferredoxin-NADPH reductase (FNR) was identified. We showed in N. tabacum and A. thaliana that a deficit of FNR1 or YCF54 caused MgProtoME accumulation, the substrate of the cyclase, and destabilization of the cyclase subunits. It is proposed that FNR serves as a potential donor for electrons required in the cyclase reaction and connects chlorophyll synthesis with photosynthetic activity., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Phosphate (P) is an essential macronutrient for plant growth. Roots employ adaptive mechanisms to forage for P in soil. Root hair elongation is particularly important since P is immobile. Here we report that auxin plays a critical role promoting root hair growth in Arabidopsis in response to low external P. Mutants disrupting auxin synthesis (taa1) and transport (aux1) attenuate the low P root hair response. Conversely, targeting AUX1 expression in lateral root cap and epidermal cells rescues this low P response in aux1. Hence auxin transport from the root apex to differentiation zone promotes auxin-dependent hair response to low P. Low external P results in induction of root hair expressed auxin-inducible transcription factors ARF19, RSL2, and RSL4. Mutants lacking these genes disrupt the low P root hair response. We conclude auxin synthesis, transport and response pathway components play critical roles regulating this low P root adaptive response.

Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Key Message: Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip ® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A. thaliana accessions. Individual probes of the GeneChip ® that show sequence polymorphisms between different A. thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A. thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A. thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

The role of phloem proteins in plant resistance to aphids is still largely elusive. By genome-wide association mapping of aphid behavior on 350 natural Arabidopsis thaliana accessions, we identified the small heat shock-like SIEVE ELEMENT-LINING CHAPERONE1 ( SLI1 ). Detailed behavioral studies on near-isogenic and knockout lines showed that SLI1 impairs phloem feeding. Depending on the haplotype, aphids displayed a different duration of salivation in the phloem. On sli1 mutants, aphids prolonged their feeding sessions and ingested phloem at a higher rate than on wild-type plants. The largest phenotypic effects were observed at 26°C, when SLI1 expression is upregulated. At this moderately high temperature, sli1 mutants suffered from retarded elongation of the inflorescence and impaired silique development. Fluorescent reporter fusions showed that SLI1 is confined to the margins of sieve elements where it lines the parietal layer and colocalizes in spherical bodies around mitochondria. This localization pattern is reminiscent of the clamp-like structures observed in previous ultrastructural studies of the phloem and shows that the parietal phloem layer plays an important role in plant resistance to aphids and heat stress., (© 2017 American Society of Plant Biologists. All rights reserved.)

Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana , the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS copurified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodeling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs. Analysis of double mutant plants defective in different combinations of TEFs revealed genetic interactions between genes encoding subunits of PAF1-C, FACT, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. Analysis of subnuclear localization, gene expression, and chromatin association did not provide evidence for an involvement of the TEFs in transcription by RNAPI (or RNAPIII). Proteomics analyses also revealed multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation, including an association of PAF1-C with the polyadenylation factor CstF. Therefore, the RNAPII transcript elongation complex represents a platform for interactions among different TEFs, as well as for coordinating ongoing transcription with mRNA processing., (© 2017 American Society of Plant Biologists. All rights reserved.)

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and diversity of centromere repeats in wild-type Arabidopsis thaliana We show that A. thaliana CENH3-containing nucleosomes exhibit a strong preference for a unique subset of centromeric repeats. These sequences are largely missing from the genome assemblies and represent the youngest and most homogeneous class of repeats. Next, we tested the evolutionary specificity of this interaction in a background in which the native A. thaliana CENH3 is replaced with CENH3s from distant species. Strikingly, we find that CENH3 from Lepidium oleraceum and Zea mays , although specifying epigenetically weaker centromeres that result in genome elimination upon outcrossing, show a binding pattern on A. thaliana centromere repeats that is indistinguishable from the native CENH3. Our results demonstrate positional stability of a highly diverged CENH3 on independently evolved repeats, suggesting that the sequence specificity of centromeres is determined by a mechanism independent of CENH3., (© 2017 Maheshwari et al.; Published by Cold Spring Harbor Laboratory Press.)

Key Message: We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

The control of genomic maintenance during S phase is crucial in eukaryotes. It involves the establishment of sister chromatid cohesion, ensuring faithful chromosome segregation, as well as proper DNA replication and repair to preserve genetic information. In animals, nuclear periphery proteins - including inner nuclear membrane proteins and nuclear pore-associated components - are key factors which regulate DNA integrity. Corresponding functional homologues are not so well known in plants which may have developed specific mechanisms due to their sessile life. We have already characterized the Gamma-tubulin Complex Protein 3-interacting proteins (GIPs) as essential regulators of centromeric cohesion at the nuclear periphery. GIPs were also shown to interact with TSA1, first described as a partner of the epigenetic regulator MGOUN3 (MGO3)/BRUSHY1 (BRU1)/TONSOKU (TSK) involved in genomic maintenance. Here, using genetic analyses, we show that the mgo3gip1 mutants display an impaired and pleiotropic development including fasciation. We also provide evidence for the contribution of both MGO3 and GIP1 to the regulation of centromeric cohesion in Arabidopsis.

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of Arabidopsis thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1 Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA., (© 2017 American Society of Plant Biologists. All rights reserved.)

Fumarate and malate are known intermediates of the TCA cycle, a mitochondrial metabolic pathway generating NADH for respiration. Arabidopsis thaliana and other Brassicaceae contain an additional cytosolic fumarase (FUM2) that functions in carbon assimilation and nitrogen use. Here, we report the identification of a hitherto unknown FUM2 promoter insertion/deletion (InDel) polymorphism found between the Col-0 and C24 accessions, which also divides a large number of Arabidopsis accessions carrying either the Col-0 or the C24 allele. The polymorphism consists of two stretches of 2.1 and 3.8 kb, which are both absent from the promotor region of Col-0 FUM2. By analysing mutants as well as mapping and natural populations with contrasting FUM2 alleles, the promotor insertion was linked to reduced FUM2 mRNA expression, reduced fumarase activity and reduced fumarate/malate ratio in leaves. In a large population of 174 natural accessions, the polymorphism was also found to be associated with the fumarate/malate ratio, malate and fumarate levels, and with dry weight at 15 days after sowing (DAS). The association with biomass production was confirmed in an even larger (251) accession population for dry weight at 22 DAS. The dominant Col-0 allele that results in increased fumarate/malate ratios and enhanced biomass production is predominantly found in central/eastern European accessions, whereas the C24 type allele is prevalent on the Iberian Peninsula, west of the Rhine and in the British Isles. Our findings support the role of FUM2 in diurnal carbon storage, and point to a growth advantage of accessions carrying the FUM2 Col-0 allele., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17-Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F-box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col-FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South-Eastern Europe approximately 5000 years ago and the FRI-Col allele to North-West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The 'American haplotype' was identified from several European localities, potentially due to return migration., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The non-photochemical quenching of light energy as heat (NPQ) is an important photoprotective mechanism that is activated in plants when light absorption exceeds the capacity of light utilization in photosynthesis. The PsbS protein plays a central role in this process and is supposed to activate NPQ through specific, light-regulated interactions with photosystem (PS) II antenna proteins. However, NPQ-specific interaction partners of PsbS in the thylakoid membrane are still unknown. Here, we have determined the localization and protein interactions of PsbS in thylakoid membranes in the NPQ-inactive (dark) and NPQ-active (light) states. Our results corroborate a localization of PsbS in PSII supercomplexes and support the model that the light activation of NPQ is based on the monomerization of dimeric PsbS and a light-induced enhanced interaction of PsbS with Lhcb1, the major component of trimeric light-harvesting complexes in PSII.

The Yang or Met Cycle is a series of reactions catalyzing the recycling of the sulfur (S) compound 5'-methylthioadenosine (MTA) to Met. MTA is produced as a by-product in ethylene, nicotianamine, and polyamine biosynthesis. Whether the Met Cycle preferentially fuels one of these pathways in a S-dependent manner remained unclear so far. We analyzed Arabidopsis (Arabidopsis thaliana) mutants with defects in the Met Cycle enzymes 5-METHYLTHIORIBOSE-1-PHOSPHATE-ISOMERASE1 (MTI1) and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (DEP1) under different S conditions and assayed the contribution of the Met Cycle to the regeneration of S for these pathways. Neither mti1 nor dep1 mutants could recycle MTA but showed S-dependent reproductive failure, which was accompanied by reduced levels of the polyamines putrescine, spermidine, and spermine in mutant inflorescences. Complementation experiments with external application of these three polyamines showed that only the triamine spermine could specifically rescue the S-dependent reproductive defects of the mutant plants. Furthermore, expressing gene-reporter fusions in Arabidopsis showed that MTI1 and DEP1 were mainly expressed in the vasculature of all plant parts. Phloem-specific reconstitution of Met Cycle activity in mti1 and dep1 mutant plants was sufficient to rescue their S-dependent mutant phenotypes. We conclude from these analyses that phloem-specific S recycling during periods of S starvation is essential for the biosynthesis of polyamines required for flowering and seed development., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The cell cycle is a complex sequence of events by which cells grow and divide mitotically or meiotically. Mitosis results in the generation of two identical daughter cells, while meiosis generates gametes as a prerequisite for sexual reproduction. To study the localization and dynamics of proteins involved in the regulation and proceeding of the cell cycle, life cell imaging of proteins fused to fluorescent tags can be performed. However, in some cases this approach cannot be applied, e.g., due to low fluorescence intensity, fast bleaching or degradation of recombinant proteins by the proteasome pathway. Instead, immunolabeling with protein-specific antibodies represents a useful approach for the analysis of intact cells. Alternatively, immunolabeling can also be applied to isolated and/or flow-sorted nuclei of particular cell cycle stages (G1, S, and G2) or of different endopolyploidy levels. This chapter details indirect immunolabeling protocols to analyze the subcellular localization and distribution of cell cycle-specific proteins in Arabidopsis thaliana.

The mitotic checkpoint (MC) guards faithful sister chromatid segregation by monitoring the attachment of spindle microtubules to the kinetochores. When chromosome attachment errors are detected, MC delays the metaphase-to-anaphase transition through the inhibition of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. In contrast to yeast and mammals, our knowledge on the proteins involved in MC in plants is scarce. Transient synchronization of root tips as well as promoter-reporter gene fusions were performed to analyze temporal and spatial expression of COPPER MODIFIED RESISTANCE1/PATRONUS1 (CMR1/PANS1) in developing Arabidopsis thaliana seedlings. Functional analysis of the gene was carried out, including CYCB1;2 stability in CMR1/PANS1 knockout and overexpressor background as well as metaphase-anaphase chromosome status. CMR1/PANS1 is transcriptionally active during M phase. Its deficiency provokes premature cell cycle exit and in consequence a rapid consumption of the number of meristematic cells in particular under stress conditions that are known to affect spindle microtubules. Root growth impairment is correlated with a failure to delay the onset of anaphase, resulting in anaphase bridges and chromosome missegregation. CMR1/PANS1 overexpression stabilizes the mitotic CYCB1;2 protein. Likely, CMR1/PANS1 coordinates mitotic cell cycle progression by acting as an APC/C inhibitor and plays a key role in growth adaptation to stress., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic protein-coding genes. Analysing the topological distribution and quantification of RNAPII can contribute to understanding its function in interphase nuclei. Previously it was shown that RNAPII molecules in plant nuclei form reticulate structures within euchromatin of differentiated Arabidopsis thaliana nuclei rather than being organized in distinct 'transcription factories' as observed in mammalian nuclei. Immunosignal intensity measurements based on specific antibody labelling in maximum intensity projections of image stacks acquired by structured illumination microscopy (SIM) suggested a relative proportional increase of RNAPII in endopolyploid plant nuclei. Here, photoactivated localization microscopy (PALM) was applied to determine the absolute number and distribution of active and inactive RNAPII molecules in differentiated A. thaliana nuclei. The proportional increase of RNAPII during endopolyploidization is confirmed, but it is also shown that PALM measurements are more reliable than those based on SIM in terms of quantification. The single molecule localization results show that, although RNAPII molecules are globally dispersed within plant euchromatin, they also aggregate within smaller distances as described for mammalian transcription factories., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The enzyme zeaxanthin epoxidase (ZEP) catalyzes the conversion of zeaxanthin to violaxanthin, a key reaction for ABA biosynthesis and the xanthophyll cycle. Both processes are important for acclimation to environmental stress conditions, in particular drought (ABA biosynthesis) and light (xanthophyll cycle) stress. Hence, both ZEP functions may require differential regulation to optimize plant fitness. The key to understanding the function of ZEP in both stress responses might lie in its spatial and temporal distribution in plant tissues. Therefore, we analyzed the distribution of ZEP in plant tissues and plastids under drought and light stress by use of a ZEP-specific antibody. In addition, we determined the pigment composition of the plant tissues and chloroplast membrane subcompartments in response to these stresses. The ZEP protein was detected in all plant tissues (except flowers) concomitant with xanthophylls. The highest levels of ZEP were present in leaf chloroplasts and root plastids. Within chloroplasts, ZEP was localized predominantly in the thylakoid membrane and stroma, while only a small fraction was bound by the envelope membrane. Light stress affected neither the accumulation nor the relative distribution of ZEP in chloroplasts, while drought stress led to an increase of ZEP in roots and to a degradation of ZEP in leaves. However, drought stress-induced increases in ABA were similar in both tissues. These data support a tissue- and stress-specific accumulation of the ZEP protein in accordance with its different functions in ABA biosynthesis and the xanthophyll cycle., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Crop plants are regularly challenged by a range of environmental stresses which typically retard their growth and ultimately compromise economic yield. The stress response involves the reprogramming of approximately 4% of the transcriptome. Here, the behavior of AtRD22 and AtUSPL1, both members of the Arabidopsis thaliana BURP (BNM2, USP, RD22 and polygalacturonase isozyme) domain-containing gene family, has been characterized. Both genes are up-regulated as part of the abscisic acid (ABA) mediated moisture stress response. While AtRD22 transcript was largely restricted to the leaf, that of AtUSPL1 was more prevalent in the root. As the loss of function of either gene increased the plant's moisture stress tolerance, the implication was that their products act to suppress the drought stress response. In addition to the known involvement of AtUSPL1 in seed development, a further role in stress tolerance was demonstrated. Based on transcriptomic data and phenotype we concluded that the enhanced moisture stress tolerance of the two loss-of-function mutants is a consequence of an enhanced basal defense response.

With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4-RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.

Seedling establishment is inhibited on media containing high levels (∼ 6%) of glucose or fructose. Genetic loci that overcome the inhibition of seedling growth on high sugar have been identified using natural variation analysis and mutant selection, providing insight into sugar signaling pathways. In this study, a quantitative trait locus (QTL) analysis was performed for seedling sensitivity to high sugar in a Col/C24 F2 population of Arabidopsis thaliana. A glucose and fructose-sensing QTL, GSQ11, was mapped through selective genotyping and confirmed in near-isogenic lines in both Col and C24 backgrounds. Allelism tests and transgenic complementation showed that GSQ11 lies within the ANAC060 gene. The Col ANAC060 allele confers sugar insensitivity and was dominant over the sugar-sensitive C24 allele. Genomic and mRNA analyses showed that a single-nucleotide polymorphism (SNP) in Col ANAC060 affects the splicing patterns of ANAC060 such that 20 additional nucleotides are present in the mRNA. The insertion created a stop codon, resulting in a truncated ANAC60 protein lacking the transmembrane domain (TMD) that is present in the C24 ANAC060 protein. The absence of the TMD results in the nuclear localization of ANAC060. The short version of the ANAC060 protein is found in ∼ 12% of natural Arabidopsis accessions. Glucose induces GSQ11/ANAC060 expression in a process that requires abscisic acid (ABA) signaling. Chromatin immunoprecipitation-qPCR and transient expression analysis showed that ABI4 directly binds to the GSQ11/ANAC060 promoter to activate transcription. Interestingly, Col ANAC060 reduced ABA sensitivity and Glc-induced ABA accumulation, and ABI4 expression was also reduced in Col ANAC060 lines. Thus, the sugar-ABA signaling cascade induces ANAC060 expression, but the truncated Col ANAC060 protein attenuates ABA induction and ABA signaling. This negative feedback from nuclear ANAC060 on ABA signaling results in sugar insensitivity.

DIGE (differential in-gel electrophoresis) is a modified version of the widely used 2-D gel electrophoresis (2-DE) for separation of complex protein samples. Two extracts to be compared are differentially labeled using fluorescent cyanine dyes and then separated together by 2-DE. An internal standard labeled using a third dye is included. This approach avoids the pitfalls of gel distortions frequently observed in the standard procedure, which hamper the subsequent gel image analysis. Inclusion of an internal standard improves the quantitative evaluation of the protein patterns. Using the advantages of the DIGE approach, impact of minor temperature differences during cold stress treatment could be quantitatively monitored. We will describe the application of DIGE to monitor the impact of cold stress on the proteome pattern of Arabidopsis. In addition to the separation of proteins, we will also outline how plant growth is performed. Finally, we will also give protocols how proteins of interest can be identified by MALDI-TOF- as well as ESI-MS/MS.

LEC1 acts as a key regulator of embryogenesis in Arabidopsis thaliana, but is involved in a wide range of functions, all the way from embryo morphogenesis to seed maturation. New data show that LEC1, partially in conjunction with abscisic acid, affects auxin synthesis, and both brassinosteroid and light signaling. The phenotype of LEC1 overexpressors confirms LEC1's known participation in the regulation of somatic embryogenesis, but also indicates additional roles in embryonic and extra-embryonic cell elongation. Here we present an integrated model of LEC1 function and suggest potential directions to be taken in future research in this important area of plant science.

The plant-specific, B3 domain-containing transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is an essential component of the regulatory network controlling the development and maturation of the Arabidopsis thaliana seed. Genome-wide chromatin immunoprecipitation (ChIP-chip), transcriptome analysis, quantitative reverse transcriptase-polymerase chain reaction and a transient promoter activation assay have been combined to identify a set of 98 ABI3 target genes. Most of these presumptive ABI3 targets require the presence of abscisic acid for their activation and are specifically expressed during seed maturation. ABI3 target promoters are enriched for G-box-like and RY-like elements. The general occurrence of these cis motifs in non-ABI3 target promoters suggests the existence of as yet unidentified regulatory signals, some of which may be associated with epigenetic control. Several members of the ABI3 regulon are also regulated by other transcription factors, including the seed-specific, B3 domain-containing FUS3 and LEC2. The data strengthen and extend the notion that ABI3 is essential for the protection of embryonic structures from desiccation and raise pertinent questions regarding the specificity of promoter recognition.