Showing papers in "The Plant Cell in 2010"
TL;DR: Despite impressive progress in understanding of the processing of Ca2+ signals during the past years, the elucidation of the exact mechanistic principles that underlie the specific recognition and conversion of the cellularCa2+ currency into defined changes in protein–protein interaction, protein phosphorylation, and gene expression and thereby establish the specificity in stimulus response coupling remain to be explored.
Abstract: Ca(2+) signals are core transducers and regulators in many adaptation and developmental processes of plants. Ca(2+) signals are represented by stimulus-specific signatures that result from the concerted action of channels, pumps, and carriers that shape temporally and spatially defined Ca(2+) elevations. Cellular Ca(2+) signals are decoded and transmitted by a toolkit of Ca(2+) binding proteins that relay this information into downstream responses. Major transduction routes of Ca(2+) signaling involve Ca(2+)-regulated kinases mediating phosphorylation events that orchestrate downstream responses or comprise regulation of gene expression via Ca(2+)-regulated transcription factors and Ca(2+)-responsive promoter elements. Here, we review some of the remarkable progress that has been made in recent years, especially in identifying critical components functioning in Ca(2+) signal transduction, both at the single-cell and multicellular level. Despite impressive progress in our understanding of the processing of Ca(2+) signals during the past years, the elucidation of the exact mechanistic principles that underlie the specific recognition and conversion of the cellular Ca(2+) currency into defined changes in protein-protein interaction, protein phosphorylation, and gene expression and thereby establish the specificity in stimulus response coupling remain to be explored.
890 citations
TL;DR: It is shown that the accumulation in barley and wheat of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis, and this may lead to an RNAi-based crop protection strategy against fungal pathogens.
Abstract: Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.
595 citations
TL;DR: Functional analysis of PYE suggests that it positively regulates growth and development under iron-deficient conditions, and Chromatin immunoprecipitation-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by regulating the expression of known ironHomeostasis genes and other genes involved in transcription, development, and stress response.
Abstract: Global population increases and climate change underscore the need for better comprehension of how plants acquire and process nutrients such as iron. Using cell type–specific transcriptional profiling, we identified a pericycle-specific iron deficiency response and a bHLH transcription factor, POPEYE (PYE), that may play an important role in this response. Functional analysis of PYE suggests that it positively regulates growth and development under iron-deficient conditions. Chromatin immunoprecipitation-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by regulating the expression of known iron homeostasis genes and other genes involved in transcription, development, and stress response. PYE interacts with PYE homologs, including IAA–Leu Resistant3 (ILR3), another bHLH transcription factor that is involved in metal ion homeostasis. Moreover, ILR3 interacts with a third protein, BRUTUS (BTS), a putative E3 ligase protein, with metal ion binding and DNA binding domains, which negatively regulates the response to iron deficiency. PYE and BTS expression is also tightly coregulated. We propose that interactions among PYE, PYE homologs, and BTS are important for maintaining iron homeostasis under low iron conditions.
513 citations
TL;DR: This work demonstrates that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth and integrates with auxin signaling to quantitatively regulate organ growth during development.
Abstract: Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.
504 citations
TL;DR: Highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice subspecies and their reciprocal hybrids reveal that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions.
Abstract: The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. Here, we report highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice (Oryza sativa) subspecies and their reciprocal hybrids. We found that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions. Differential epigenetic modifications correlated with changes in transcript levels among hybrids and parental lines. Distinct patterns in gene expression and epigenetic modifications in reciprocal hybrids were observed. Through analyses of single nucleotide polymorphisms from our sequence data, we observed a high correlation of allelic bias of epigenetic modifications or gene expression in reciprocal hybrids with their differences in the parental lines. The abundance of distinct small RNA size classes differed between the parents, and more small RNAs were downregulated than upregulated in the reciprocal hybrids. Together, our data reveal a comprehensive overview of transcriptional and epigenetic trends in heterotic rice crosses and provide a useful resource for the rice community.
503 citations
TL;DR: These experiments demonstrate the presence of highly orchestrated and tissue-specific MAMP responses in roots and potential pathogen-encoded mechanisms to block these MAMP-elicited signaling pathways.
Abstract: Despite the fact that roots are the organs most subject to microbial interactions, very little is known about the response of roots to microbe-associated molecular patterns (MAMPs). By monitoring transcriptional activation of β-glucuronidase reporters and MAMP-elicited callose deposition, we show that three MAMPs, the flagellar peptide Flg22, peptidoglycan, and chitin, trigger a strong tissue-specific response in Arabidopsis thaliana roots, either at the elongation zone for Flg22 and peptidoglycan or in the mature parts of the roots for chitin. Ethylene signaling, the 4-methoxy-indole-3-ylmethylglucosinolate biosynthetic pathway, and the PEN2 myrosinase, but not salicylic acid or jasmonic acid signaling, play major roles in this MAMP response. We also show that Flg22 induces the cytochrome P450 CYP71A12-dependent exudation of the phytoalexin camalexin by Arabidopsis roots. The phytotoxin coronatine, an Ile-jasmonic acid mimic produced by Pseudomonas syringae pathovars, suppresses MAMP-activated responses in the roots. This suppression requires the E3 ubiquitin ligase COI1 as well as the transcription factor JIN1/MYC2 but does not rely on salicylic acid–jasmonic acid antagonism. These experiments demonstrate the presence of highly orchestrated and tissue-specific MAMP responses in roots and potential pathogen-encoded mechanisms to block these MAMP-elicited signaling pathways.
491 citations
TL;DR: The results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.
Abstract: An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.
488 citations
TL;DR: A group of WRKY transcription factors that function as negative abscisic acid (ABA) signaling regulators acting as transcription repressors directly downstream of a previously identified ABA receptor ABAR are identified, describing an ABA signaling pathway from primary events to downstream gene expression.
Abstract: The phytohormone abscisic acid (ABA) plays a vital role in plant development and response to environmental challenges, but the complex networks of ABA signaling pathways are poorly understood. We previously reported that a chloroplast protein, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), functions as a receptor for ABA in Arabidopsis thaliana. Here, we report that ABAR spans the chloroplast envelope and that the cytosolic C terminus of ABAR interacts with a group of WRKY transcription factors (WRKY40, WRKY18, and WRKY60) that function as negative regulators of ABA signaling in seed germination and postgermination growth. WRKY40, a central negative regulator, inhibits expression of ABA-responsive genes, such as ABI5. In response to a high level of ABA signal that recruits WRKY40 from the nucleus to the cytosol and promotes ABAR-WRKY40 interaction, ABAR relieves the ABI5 gene of inhibition by repressing WRKY40 expression. These findings describe a unique ABA signaling pathway from the early signaling events to downstream gene expression.
466 citations
TL;DR: Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and R DR6.
Abstract: Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins.
464 citations
TL;DR: Analysis of the NC64A genome substantially advances the understanding of the green lineage evolution, including the genomic interplay with viruses and symbiosis between eukaryotes.
Abstract: Chlorella variabilis NC64A, a unicellular photosynthetic green alga (Trebouxiophyceae), is an intracellular photobiont of Paramecium bursaria and a model system for studying virus/algal interactions. We sequenced its 46-Mb nuclear genome, revealing an expansion of protein families that could have participated in adaptation to symbiosis. NC64A exhibits variations in GC content across its genome that correlate with global expression level, average intron size, and codon usage bias. Although Chlorella species have been assumed to be asexual and nonmotile, the NC64A genome encodes all the known meiosis-specific proteins and a subset of proteins found in flagella. We hypothesize that Chlorella might have retained a flagella-derived structure that could be involved in sexual reproduction. Furthermore, a survey of phytohormone pathways in chlorophyte algae identified algal orthologs of Arabidopsis thaliana genes involved in hormone biosynthesis and signaling, suggesting that these functions were established prior to the evolution of land plants. We show that the ability of Chlorella to produce chitinous cell walls likely resulted from the capture of metabolic genes by horizontal gene transfer from algal viruses, prokaryotes, or fungi. Analysis of the NC64A genome substantially advances our understanding of the green lineage evolution, including the genomic interplay with viruses and symbiosis between eukaryotes.
457 citations
TL;DR: It is demonstrated that, through perception of Peps, PEPR1 and PEPR2 contribute to defense responses in Arabidopsis, and differential binding affinities of two receptors with a family of peptide ligands are demonstrated.
Abstract: Pep1 is a 23–amino acid peptide that enhances resistance to a root pathogen, Pythium irregulare. Pep1 and its homologs (Pep2 to Pep7) are endogenous amplifiers of innate immunity of Arabidopsis thaliana that induce the transcription of defense-related genes and bind to PEPR1, a plasma membrane leucine-rich repeat (LRR) receptor kinase. Here, we identify a plasma membrane LRR receptor kinase, designated PEPR2, that has 76% amino acid similarity to PEPR1, and we characterize its role in the perception of Pep peptides and defense responses. Both PEPR1 and PEPR2 were transcriptionally induced by wounding, treatment with methyl jasmonate, Pep peptides, and pathogen-associated molecular patterns. The effects of Pep1 application on defense-related gene induction and enhancement of resistance to Pseudomonas syringae pv tomato DC3000 were partially reduced in single mutants of PEPR1 and PEPR2 and abolished completely in double mutants. Photoaffinity labeling and binding assays using transgenic tobacco (Nicotiana tabacum) cells expressing PEPR1 and PEPR2 clearly demonstrated that PEPR1 is a receptor for Pep1-6 and that PEPR2 is a receptor for Pep1 and Pep2. Our analysis demonstrates differential binding affinities of two receptors with a family of peptide ligands and the corresponding physiological effects of the specific receptor–ligand interactions. Therefore, we demonstrate that, through perception of Peps, PEPR1 and PEPR2 contribute to defense responses in Arabidopsis.
TL;DR: Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal, suggesting at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells.
Abstract: The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell-cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.
TL;DR: Examination of secretory and endocytotic trafficking in Arabidopsis provides evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrates that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation.
Abstract: Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.
TL;DR: It is demonstrated that AP2 has dual molecular roles, directly inducing the expression of the floral repressor AGAMOUS-LIKE15 and directly repressing the transcription of floral activators like SUPPRESSOR of OVEREXPRESSION of CONSTANS1.
Abstract: The Arabidopsis thaliana transcription factor APETALA2 (AP2) has numerous functions, including roles in seed development, stem cell maintenance, and specification of floral organ identity. To understand the relationship between these different roles, we mapped direct targets of AP2 on a genome-wide scale in two tissue types. We find that AP2 binds to thousands of loci in the developing flower, many of which exhibit AP2-dependent transcription. Opposing, logical effects are evident in AP2 binding to two microRNA genes that influence AP2 expression, with AP2 positively regulating miR156 and negatively regulating miR172, forming a complex direct feedback loop, which also included all but one of the AP2-like miR172 target clade members. We compare the genome-wide direct target repertoire of AP2 with that of SCHLAFMUTZE, a closely related transcription factor that also represses the transition to flowering. We detect clear similarities and important differences in the direct target repertoires that are also tissue specific. Finally, using an inducible expression system, we demonstrate that AP2 has dual molecular roles. It functions as both a transcriptional activator and repressor, directly inducing the expression of the floral repressor AGAMOUS-LIKE15 and directly repressing the transcription of floral activators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1.
TL;DR: In vivo involvement of PDAT1 in TAG biosynthesis is established, major contributions by other candidate enzymes are ruled out, and overlapping functions that are essential for normal pollen and seed development of Arabidopsis are indicated.
Abstract: Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.
TL;DR: It is demonstrated that ethylene similarly regulates EIN3 and EIL1, the two master transcription factors coordinating myriad ethylene responses, and it is clarified that EIN2 but not MKK9 is required for ethylene-induced Ein3/EIL1 stabilization.
Abstract: Plant responses to ethylene are mediated by regulation of EBF1/2-dependent degradation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Here, we report that the level of EIL1 protein is upregulated by ethylene through an EBF1/2-dependent pathway. Genetic analysis revealed that EIL1 and EIN3 cooperatively but differentially regulate a wide array of ethylene responses, with EIL1 mainly inhibiting leaf expansion and stem elongation in adult plants and EIN3 largely regulating a multitude of ethylene responses in seedlings. When EBF1 and EBF2 are disrupted, EIL1 and EIN3 constitutively accumulate in the nucleus and remain unresponsive to exogenous ethylene application. Further study revealed that the levels of EBF1 and EBF2 proteins are downregulated by ethylene and upregulated by silver ion and MG132, suggesting that ethylene stabilizes EIN3/EIL1 by promoting EBF1 and EBF2 proteasomal degradation. Also, we found that EIN2 is indispensable for mediating ethylene-induced EIN3/EIL1 accumulation and EBF1/2 degradation, whereas MKK9 is not required for ethylene signal transduction, contrary to a previous report. Together, our studies demonstrate that ethylene similarly regulates EIN3 and EIL1, the two master transcription factors coordinating myriad ethylene responses, and clarify that EIN2 but not MKK9 is required for ethylene-induced EIN3/EIL1 stabilization. Our results also reveal that EBF1 and EBF2 act as essential ethylene signal transducers that by themselves are subject to proteasomal degradation.
TL;DR: Variations in the TAL effector repetitive domains are driven by selection to overcome both dominant and recessive forms of resistance to bacterial blight in rice, indicating that N3 proteins have a specific function in facilitating bacterial blight disease.
Abstract: The rice (Oryza sativa) gene xa13 is a recessive resistance allele of Os-8N3, a member of the NODULIN3 (N3) gene family, located on rice chromosome 8. Os-8N3 is a susceptibility (S) gene for Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight, and the recessive allele is defeated by strains of the pathogen producing any one of the type III effectors AvrXa7, PthXo2, or PthXo3, which are all members of the transcription activator-like (TAL) effector family. Both AvrXa7 and PthXo3 induce the expression of a second member of the N3 gene family, here named Os-11N3. Insertional mutagenesis or RNA-mediated silencing of Os-11N3 resulted in plants with loss of susceptibility specifically to strains of X. oryzae pv oryzae dependent on AvrXa7 or PthXo3 for virulence. We further show that AvrXa7 drives expression of Os-11N3 and that AvrXa7 interacts and binds specifically to an effector binding element within the Os-11N3 promoter, lending support to the predictive models for TAL effector binding specificity. The result indicates that variations in the TAL effector repetitive domains are driven by selection to overcome both dominant and recessive forms of resistance to bacterial blight in rice. The finding that Os-8N3 and Os-11N3 encode closely related proteins also provides evidence that N3 proteins have a specific function in facilitating bacterial blight disease.
TL;DR: It is demonstrated that NRAMP1 is a physiological manganese transporter in Arabidopsis and restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating thatNRAMP1 has a broad selectivity in vivo.
Abstract: In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis.
TL;DR: In this paper, the authors identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH.
Abstract: Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.
TL;DR: The results provide evidence that environmental conditions can override epigenetic regulation, at least transiently, which might open a window for more permanent epigenetic changes.
Abstract: Epigenetic factors determine responses to internal and external stimuli in eukaryotic organisms. Whether and how environmental conditions feed back to the epigenetic landscape is more a matter of suggestion than of substantiation. Plants are suitable organisms with which to address this question due to their sessile lifestyle and diversification of epigenetic regulators. We show that several repetitive elements of Arabidopsis thaliana that are under epigenetic regulation by transcriptional gene silencing at ambient temperatures and upon short term heat exposure become activated by prolonged heat stress. Activation can occur without loss of DNA methylation and with only minor changes to histone modifications but is accompanied by loss of nucleosomes and by heterochromatin decondensation. Whereas decondensation persists, nucleosome loading and transcriptional silencing are restored upon recovery from heat stress but are delayed in mutants with impaired chromatin assembly functions. The results provide evidence that environmental conditions can override epigenetic regulation, at least transiently, which might open a window for more permanent epigenetic changes.
TL;DR: The endoplasmic reticulum has a sophisticated quality control (QC) system to eliminate improperly folded proteins from the secretory pathway, but the extent to which this system contributes to various stress responses in plants is unclear.
Abstract: The endoplasmic reticulum (ER) has a sophisticated quality control (QC) system to eliminate improperly folded proteins from the secretory pathway. Given that protein folding is such a fastidious process and subject to adverse environmental conditions, the ER QC system appears to have been usurped to serve as an environmental sensor and responder in plants. Under stressful conditions, the ER protein folding machinery reaches a limit as the demands for protein folding exceed the capacity of the system. Under these conditions, misfolded or unfolded proteins accumulate in the ER, triggering an unfolded protein response (UPR). UPR mitigates ER stress by upregulating the expression of genes encoding components of the protein folding machinery or the ER-associated degradation system. In Arabidopsis thaliana, ER stress is sensed and stress signals are transduced by membrane-bound transcription factors, which are activated and mobilized under environmental stress conditions. Under acute or chronic stress conditions, UPR can also lead to apoptosis or programmed cell death. Despite recent progress in our understanding of plant protein QC, discovering how different environmental conditions are perceived is one of the major challenges in understanding this system. Since the ER QC system is one among many stress response systems in plants, another major challenge is determining the extent to which the ER QC system contributes to various stress responses in plants.
TL;DR: The results demonstrate a conserved mechanism of BR regulation of plant development through a pair of antagonizing HLH/bHLH transcription factors that act downstream of BZR1 in Arabidopsis and rice.
Abstract: In rice (Oryza sativa), brassinosteroids (BRs) induce cell elongation at the adaxial side of the lamina joint to promote leaf bending. We identified a rice mutant (ili1-D) showing an increased lamina inclination phenotype similar to that caused by BR treatment. The ili1-D mutant overexpresses an HLH protein homologous to Arabidopsis thaliana Paclobutrazol Resistance1 (PRE1) and the human Inhibitor of DNA binding proteins. Overexpression and RNA interference suppression of ILI1 increase and reduce, respectively, rice laminar inclination, confirming a positive role of ILI1 in leaf bending. ILI1 and PRE1 interact with basic helix-loop-helix (bHLH) protein IBH1 (ILI1 binding bHLH), whose overexpression causes erect leaf in rice and dwarfism in Arabidopsis. Overexpression of ILI1 or PRE1 increases cell elongation and suppresses dwarf phenotypes caused by overexpression of IBH1 in Arabidopsis. Thus, ILI1 and PRE1 may inactivate inhibitory bHLH transcription factors through heterodimerization. BR increases the RNA levels of ILI1 and PRE1 but represses IBH1 through the transcription factor BZR1. The spatial and temporal expression patterns support roles of ILI1 in laminar joint bending and PRE1/At IBH1 in the transition from growth of young organs to growth arrest. These results demonstrate a conserved mechanism of BR regulation of plant development through a pair of antagonizing HLH/bHLH transcription factors that act downstream of BZR1 in Arabidopsis and rice.
TL;DR: This review presents general features of plant and animal AGO proteins and detail the authors' knowledge on the 10 Arabidopsis thaliana AGOs.
Abstract: Both transcriptional (TGS) and posttranscriptional gene silencing (PTGS) are conserved eukaryotic gene regulatory mechanisms, integral for taming exogenous (viruses and bacteria) or endogenous (repetitive elements and transposons) invasive nucleic acids to minimize their impact on genome integrity and function. TGS and PTGS also are essential for controlling the expression of protein coding genes throughout development or in response to environmental stimuli. In plants and animals, at least one member of the conserved ARGONAUTE (AGO) protein family comprises the catalytic engine of the silencing complex, which is guided by sequence-specific small RNA to cognate RNA. In this review, we present general features of plant and animal AGO proteins and detail our knowledge on the 10 Arabidopsis thaliana AGOs.
TL;DR: The results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.
Abstract: Reactive oxygen species and redox signaling undergo synergistic and antagonistic interactions with phytohormones to regulate protective responses of plants against biotic and abiotic stresses. However, molecular insight into the nature of this crosstalk remains scarce. We demonstrate that the hydrogen peroxide-responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). Biochemical characterization of recombinant UGT74E2 demonstrated that it strongly favors IBA as a substrate. Assessment of indole-3-acetic acid (IAA), IBA, and their conjugates in transgenic plants ectopically expressing UGT74E2 indicated that the catalytic specificity was maintained in planta. In these transgenic plants, not only were IBA-Glc concentrations increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This perturbed IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.
TL;DR: The genetic underpinnings of an emerging phenotypic model where wheat domestication has transformed a long thin primitive grain to a wider and shorter modern grain are provided.
Abstract: Grain morphology in wheat (Triticum aestivum) has been selected and manipulated even in very early agrarian societies and remains a major breeding target. We undertook a large-scale quantitative analysis to determine the genetic basis of the phenotypic diversity in wheat grain morphology. A high-throughput method was used to capture grain size and shape variation in multiple mapping populations, elite varieties, and a broad collection of ancestral wheat species. This analysis reveals that grain size and shape are largely independent traits in both primitive wheat and in modern varieties. This phenotypic structure was retained across the mapping populations studied, suggesting that these traits are under the control of a limited number of discrete genetic components. We identified the underlying genes as quantitative trait loci that are distinct for grain size and shape and are largely shared between the different mapping populations. Moreover, our results show a significant reduction of phenotypic variation in grain shape in the modern germplasm pool compared with the ancestral wheat species, probably as a result of a relatively recent bottleneck. Therefore, this study provides the genetic underpinnings of an emerging phenotypic model where wheat domestication has transformed a long thin primitive grain to a wider and shorter modern grain.
TL;DR: This research demonstrates that NO acts as a redox regulator of a transcription factor network that is a key component of plant defense and systemic acquired resistance and suggests that nitric oxide is a red ox regulator of the NPR1/TGA1 system.
Abstract: The role of reactive oxygen and nitrogen species in local and systemic defense reactions is well documented. NPR1 and TGA1 are key redox-controlled regulators of systemic acquired resistance in plants. NPR1 monomers interact with the reduced form of TGA1, which targets the activation sequence-1 (as-1) element of the promoter region of defense proteins. Here, we report the effect of the physiological nitric oxide donor S-nitrosoglutathione on the NPR1/TGA1 regulation system in Arabidopsis thaliana. Using the biotin switch method, we demonstrate that both NPR1 and TGA1 are S-nitrosylated after treatment with S-nitrosoglutathione. Mass spectrometry analyses revealed that the Cys residues 260 and 266 of TGA1 are S-nitrosylated and S-glutathionylated even at GSNO concentrations in the low micromolar range. Furthermore, we showed that S-nitrosoglutathione protects TGA1 from oxygen-mediated modifications and enhances the DNA binding activity of TGA1 to the as-1 element in the presence of NPR1. In addition, we observed that the translocation of NPR1 into the nucleus is promoted by nitric oxide. Taken together, our results suggest that nitric oxide is a redox regulator of the NPR1/TGA1 system and that they underline the importance of nitric oxide in the plant defense response.
TL;DR: It is shown that sRNA length and 5′ nucleotide do not account for the observed functional diversification of these AGOs, and the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications is highlighted.
Abstract: Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5' adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5' nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications.
TL;DR: This work provides evidence that both bZIP28 and NF-Y subunits are recruited under stress to form a transcriptional complex that upregulates the expression of endoplasmic reticulum stress-induced genes.
Abstract: Stress agents known to elicit the unfolded protein response in Arabidopsis thaliana upregulate the expression of a constellation of genes dependent on the membrane-associated basic domain/leucine zipper (bZIP) transcription factor, bZIP28. Among the stress-activated genes, a consensus promoter sequence corresponding to the endoplasmic reticulum (ER) stress-responsive element I (ERSE-I), CCAAT-N10-CACG, was identified. Disruption of either the CCAAT or CACG subelement in ERSE-I resulted in reduction of the transcriptional response to ER stress. bZIP28 forms homo- and heterodimers with other bZIP TF family members (in subgroup D) and interacts with CCAAT box binding factors, heterotrimeric factors composed of NF-Y subunits. Arabidopsis encodes 36 NF-Y subunits, and it was found that subunits NF-YB3 and -YC2 interact with bZIP28 and NF-YA4, respectively, in a yeast three-hybrid system. A transcriptional complex containing bZIP28 and the above-mentioned three NF-Y subunits was assembled in vitro on DNA containing ERSE-I. bZIP28, on its own, binds to the CACG subelement in ERSE-I to form a smaller complex I, and in combination with the NF-Y subunits above, bZIP28 assembles into a larger transcriptional complex (complex II). bZIP28 was shown to interact with NF-Y subunits in vivo in bimolecular fluorescence complementation analyses and in coimmunoprecipitation assays. Treatment of seedlings with ER stress agents led to the upregulation of NF-YC2 and the relocation of NF-YB3 from the cytoplasm to the nucleus. Thus, in response to ER stress, bZIP28 is mobilized by proteolysis and recruits NF-Y subunits to form a transcriptional complex that upregulates the expression of ER stress-induced genes.
TL;DR: The functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana, suggests that NRT 1.8-regulated nitrate distribution plays an important role in Cd2+ tolerance.
Abstract: Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-beta-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd(2+)) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd(2+)-sensitive phenotype. Further analyses showed that Cd(2+) stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd(2+) tolerance.
TL;DR: It is proposed that evolutionarily diverse CIN-like TCPs have important roles in the signaling pathways that generate different leaf forms, without having any lethal effects on shoots.
Abstract: Coordination of the maintenance of the undifferentiated fate of cells in the shoot meristem and the promotion of cellular differentiation in plant organs is essential for the development of plant shoots. CINCINNATA-like (CIN-like) TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors are involved in this coordination via the negative regulation of CUP-SHAPED COTYLEDON (CUC) genes, which regulate the formation of shoot meristems and the specification of organ boundaries. However, the molecular mechanism of the action of CIN-like TCPs is poorly understood. We show here that TCP3, a model of CIN-like TCPs of Arabidopsis thaliana, directly activates the expression of genes for miR164, ASYMMETRIC LEAVES1 (AS1), INDOLE-3-ACETIC ACID3/SHORT HYPOCOTYL2 (IAA3/SHY2), and SMALL AUXIN UP RNA (SAUR) proteins. Gain of function of these genes suppressed the formation of shoot meristems and resulted in the fusion of cotyledons, whereas their loss of function induced ectopic expression of CUC genes in leaves. Our results indicate that miR164, AS1, IAA3/SHY2, and SAUR partially but cooperatively suppress the expression of CUC genes. Since CIN-like TCP genes were revealed to act dose dependently in the differentiation of leaves, we propose that evolutionarily diverse CIN-like TCPs have important roles in the signaling pathways that generate different leaf forms, without having any lethal effects on shoots.