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Abstract: The Arabidopsis paraquat resistant1 (PAR1) was classified as L-type amino acid transporter 4 (LAT4) based on a phylogenetic analysis of selected genes from Saccharomyces cerevisiae, Arabidopsis thaliana (L.) Heynh, [...]

Posttranslational modifications of histones play important roles in modulating chromatin structure and regulating gene expression. We have previously shown that more than two thirds of Arabidopsis genes contain histone H3 methylation at lysine 4 (H3K4me) and that trimethylation of H3K4 (H3K4me3) is preferentially located at actively transcribed genes. In addition, several Arabidopsis mutants with locus-specific loss of H3K4me have been found to display various developmental abnormalities. These findings suggest that H3K4me3 may play important roles in maintaining the normal expression of a large number of genes. However, the major enzyme(s) responsible for H3K4me3 has yet to be identified in plants, making it difficult to address questions regarding the mechanisms and functions of H3K4me3. Here we described the characterization of SET DOMAIN GROUP 2 (SDG2), a large Arabidopsis protein containing a histone lysine methyltransferase domain. We found that SDG2 hom01ogs are highly conserved in plants and that the Arabidopsis SDG2 gene is broadly expressed during development. In addition, the loss of SDG2 leads to severe and pleiotropic phenotypes, as well as the misregulation of a large number of genes. Consistent with our finding that SDG2 is a robust and specific H3K4 methyltransferase in vitro, the loss of SDG2 leads to a drastic decrease in H3K4me3 in vivo. Taken together, these results suggest that SDG2 is the major enzyme responsible for H3K4me3 in Arabidopsis and that SDG2-dependent H3K4m3 is critical for regulating gene expression and plant development. chromatin | methylation doi/ 10.1073/pnas.1010478107

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP, plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals. Arabidopsis | Drosophila | organ development | cell division www.pnas.org/cgi/doi/ 10.1073/pnas.1007926107

Plants and animals produce modular developmental units in a periodic fashion. In plants, lateral roots form as repeating units along the root primary axis; however, the developmental mechanism regulating this process is unknown. We found that cyclic expression pulses of a reporter gene mark the position of future Lateral roots by establishing prebranch sites and that prebranch site production and root bending are periodic. Microarray and promoter-luciferase studies revealed two sets of genes oscillating in opposite phases at the root tip. Genetic studies show that some oscillating transcriptional regulators are required for periodicity in one or both developmental processes. This molecular mechanism has characteristics that resemble molecular clock--driven activities in animal species. 10.1126/science.1191937

In plant innate immunity, the leucine-rich repeat receptor kinase FLS2 recognizes the bacterial pathogen-associated molecular pattern (PAMP) flagellin. The molecular mechanisms underlying PAMP perception are not fully understood. Here, we reveal that the gaseous phytohormone ethylene is an integral part of PAMP-triggered immunity. Plants mutated in the key ethylene-signaling protein EIN2 are impaired in all FLS2-mediated responses, correlating with reduced FLS2 transcription and protein accumulation. The EIN3 and EIN3-like transcription factors, which depend on EIN2 activity for their accumulation, directly control FLS2 expression. Our results reveal a direct role for ethylene in regulation of an innate immune receptor. innate immunity | receptor kinase doi/ 10.1073/pnas.1003347107

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive 1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites. PDR9 | PDR8 | IBA transport | auxin synthesis doi/ 10.1073/pnas.1007107

An imbalance in photosynthetic electron transfer is thought to be redressed by photosynthetic control of the rate of expression of genes encoding apoproteins of photosystem (PS)-I and PS-II in response to the redox state of plastoquinone (PQ), which is a connecting electron carrier. PS stoichiometry is then adjusted to enhance photosynthetic efficiency. In prokaryotes, sigma factors are well known for their participation in the control of RNA polymerase activity in transcription, whereas there have been no reports concerning their association with redox regulation. We have found that the phosphorylation of SIG1, the major sigma factor (SIG), is regulated by redox signals and selectively inhibits the transcription of the psaA gene, which encodes a PS-I protein. We produced transgenic Arabidopsis plants with or without the putative phosphorylation sites for SIG1 and demonstrated through in vivo labeling that Thr-170 was involved in the phosphorylation. We analyzed the in vivo and in vitro transcriptional responses of the transgenic Arabidopsis plants to the redox status in regard to involvement of the phosphorylation site. We revealed an enhanced phosphorylation of SIG1 under oxidative conditions of PQ in a form associated with the molecular mass of the holoenzyme. Phosphorylation of SIG1 proved crucial through a change in the promoter specificity for sustaining balanced expression of components in PS-I and PS-II and was responsible for harmonious electron flow to maintain photosynthetic efficiency. chloroplast | redox regulation | plastoquinone | RNA polymerase | Arabidopsis doi/ 10.1073/pnas.0911692107

Oligogalacturonides (OGs) released from the plant cell wall are active both as damage-associated molecular patterns (DAMPs) for the activation of the plant immune response and regulators of plant growth and development. Members of the Wall-Associated Kinase (WAK) family are candidate receptors of OGs, due to their ability to bind in vitro these oligosaccharides. Because lethality and redundancy have hampered the study of WAKs by reverse genetics, we have adopted a chimeric receptor approach to elucidate the role of Arabidopsis WAK1. In a test-of-concept study, we first defined the appropriate chimera design and demonstrated that the Arabidopsis pattern recognition receptor (PRR) EFR is amenable to the construction of functional and resistance-conferring chimeric receptors carrying the ectodomain of another Arabidopsis PRR, FLS2. After, we analyzed chimeras derived from EFR and WAK1. Our results show that, upon stimulation with OGs, the WAK1 ectodomain is capable of activating the EFR kinase domain. On the other hand, upon stimulation with the cognate ligand elf18, the EFR ectodomain activates the WAK1 kinase, triggering defense responses that mirror those normally activated by OGs and are effective against fungal and bacterial pathogens. Finally, we show that transgenic plants overexpressing WAK1 are more resistant to Botrytis cinerea. damage-associated molecular patterns | elongation factor tu receptor | pectin-mediated signaling | plant immunity | chimeric receptors doi: 10.1073/pnas.1000675107

Quantitative trait loci analysis of natural Arabidopsis thaliana accessions is increasingly exploited for gene isolation. However, to date this has mostly revealed deleterious mutations. Among them, a loss-of-function allele identified the root growth regulator BREVIS RADIX (BRX). Here we present evidence that BRX and the paralogous BRX-LIKE (BRXL) genes are under selective constraint in monocotyledons as well as dicotyledons. Unexpectedly, however, whereas none of the Arabidopsis orthologs except AtBRXL1 could complement brx null mutants when expressed constitutively, nearly all monocotyledon BRXLs tested could. Thus, BRXL proteins seem to be more diversified in dicotyledons than in monocotyledons. This functional diversification was correlated with accelerated rates of sequence divergence in the N-terminal regions. Population genetic analyses of 30 haplotypes are suggestive of an adaptive role of AtBRX and AtBRXL1. In two accessions, Lc-0 and Lov-5, seven amino acids are deleted in the variable region between the highly conserved C-terminal, so-called BRX domains. Genotyping of 42 additional accessions also found this deletion in Kz-1, Pu2-7, and Ws-0. In segregating recombinant inbred lines, the Lc-0 allele ([AtBRX.sup.Lc-O]) conferred significantly enhanced root growth. Moreover, when constitutively expressed in the same regulatory context, [AtBRX.sup.Lc-O] complemented brx mutants more efficiently than an allele without deletion. The same was observed for AtBRXL1, which compared with AtBRX carries a 13 amino acid deletion that encompasses the deletion found in [AtBRX.sup.Lc-O]. Thus, the [AtBRX.sup.Lc-O] allele seems to contribute to natural variation in root growth vigor and provides a rare example of an experimentally confirmed, hyperactive allelic variant. Brachypodium | BREVIS RADIX | adaptation doi/ 10.1073/pnas.0913207107

The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiting and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. Although AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning, and hormonal pathways. 10.1126/science.1185244

Digestion-ligation-amplification (DLA), a novel adaptor-mediated PCR-based method that uses a single-stranded oligo as the adaptor, was developed to overcome difficulties of amplifying unknown sequences flanking known DNA sequences in large genomes. DLA specifically overcomes the problems associated with existing methods for amplifying genomic sequences flanking Mu transposons, including high levels of nonspecific amplification. Two DLA-based strategies, MuClone and DLA-454, were developed to isolate Mu-tagged alleles. MuClone allows for the amplification of subsets of the numerous Mu transposons in the genome, using unique three-nucleotide tags at the 3? ends of primers, simplifying the identification of flanking sequences that cosegregate with mutant phenotypes caused by Mu insertions. DLA-454, which combines DLA with 454 pyrosequencing, permits the efficient cloning of genes for which multiple independent insertion alleles are available without the need to develop segregating populations. The utility of each approach was validated by independently cloning the gl4 (glossy4) gene. Mutants of gl4 lack the normal accumulation of epicuticular waxes. The gl4 gene is a homolog of the Arabidopsis CUT1 gene, which encodes a condensing enzyme involved in the synthesis of very-long-chain fatty acids, which are precursors of epicuticular waxes.

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA)--guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)--miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/ miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4-6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing. high-throughput sequencing | miRNA | trans-acting siRNA www.pnas.org/cgi/doi/10.1073/pnas.0913203107

Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion ceils of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels. www.pnas.org/cgi/doi/10.1073/pnas.0906131106

The polyunsaturated fatty acids (PUFAs) linoleic acid (18:2) and [alpha]-Iinolenic acid (18:3) in triacylglycerols (TAG) are major factors affecting the quality of plant oils for human health, as well as for biofuels and other renewable applications. These PUFAs are essential fatty acids for animals and plants, but also are the source of unhealthy trans fats during the processing of many foodstuffs. PUFAs 18:2 and 18:3 are synthesized in developing seeds by the desaturation of oleic acid (18:1) esterified on the membrane lipid phosphatidylcholine (PC) on the endoplasmic reticulum. The reactions and fluxes involved in this metabolism are incompletely understood, however. Here we show that a previously unrecognized enzyme, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), encoded by the Arabidopsis ROD1 gene, is a major reaction for the transfer of 18:1 into PC for desaturation and also for the reverse transfer of 18:2 and 18:3 into the TAG synthesis pathway. The PDCT enzyme catalyzes transfer of the phosphocholine headgroup from PC to diacylglycerol, and mutation of rod1 reduces 18:2 and 18:3 accumulation in seed TAG by 40%. Our discovery of PDCT is important for understanding glycerolipid metabolism in plants and other organisms, and provides tools to modify the fatty acid compositions of plant oils for improved nutrition, biofuel, and other purposes. Arabidopsis | lipid metabolism | oilseeds www.pnas.org/cgi/doi/10.1073/pnas.0909731106

Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis. We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10-18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination. genome maintenance | microhomology | recombination

The Arabidopsis sog1-1 (suppressor of gamma response) mutant was originally isolated as a second-site suppressor of the radiosensitive phenotype of seeds defective in the repair endonuclease XPF. Here, we report that SOG1 encodes a putative transcription factor. This gene is a member of the NAC domain [petunia NAM (no apical meristem) and Arabidopsis ATAF1,2 and CUC2] family (a family of proteins unique to land plants). Hundreds of genes are normally up-regulated in Arabidopsis within an hour of treatment with ionizing radiation; the induction of these genes requires the damage response protein kinase ATM, but not the related kinase ATR. Here, we find that SOG1 is also required for this transcriptional up-regulation. In contrast, the SOGl-dependent checkpoint response observed in xpfmutant seeds requires ATR, but does not require ATM. Thus, phenotype of the sog1-1 mutant mimics aspects of the phenotypes of both atr and atm mutants in Arabidopsis, suggesting that SOG1 participates in pathways governed by both of these sensor kinases. We propose that, in plants, signals related to genomic stress are processed through a single, central transcription factor, SOG1. Arabidopsis | ATM | ATR |checkpoint response | XPF

Plant organs originate from meristems where stem cells are maintained to produce continuously daughter cells that are the source of different cell types. The cell cycle switch gene CCS52A, a substrate specific activator of the anaphase promoting complex/ cyclosome (APC/C), controls the mitotic arrest and the transition of mitotic cycles to endoreduplication (ER) cycles as part of cell differentiation. Arabidopsis, unlike other organisms, contains 2 CCS52A isoforms. Here, we show that both of them are active and regulate meristem maintenance in the root tip, although through different mechanisms. The CCS52A1 activity in the elongation zone of the root stimulates ER and mitotic exit, and contributes to the border delineation between dividing and expanding cells. In contrast, CCS52A2 acts directly in the distal region of the root meristem to control identity of the quiescent center (QC) cells and stem cell maintenance. Cell proliferation assays in roots suggest that this control involves CCS52A2 mediated repression of mitotic activity in the QC cells. The data indicate that the CCS52A genes favor a low mitotic state in different cell types of the root tip that is required for meristem maintenance, and reveal a previously undescribed mechanism for APC/C mediated control in plant development. CDH1 | cell differentiation | endoreduplication | quiescent center | stem cells

Both plants and animals require the activity of proteins containing nucleotide binding (NB) domain and leucine-rich repeat (LRR) domains for proper immune system function. NB-LRR proteins in plants (NLR proteins in animals) also require conserved regulation via the proteins SGT1 and cytosolic HSP90. RAR1, a protein specifically required for plant innate immunity, interacts with SGT1 and HSP90 to maintain proper NB-LRR protein steady-state levels. Here, we present the identification and characterization of specific mutations in Arabidopsis HSP90.2 that suppress all known phenotypes of rar1. These mutations are unique with respect to the many mutant alleles of HSP90 identified in all systems in that they can bypass the requirement for a cochaperone and result in the recovery of client protein accumulation and function. Additionally, these mutations separate HSP90 ATP hydrolysis from HSP90 function in client protein folding and/or accumulation. By recapitulating the activity of RAR1, these novel hsp90 alleles allow us to propose that RAR1 regulates the physical open-close cycling of a known 'lid structure' that is used as a dynamic regulatory HSP90 mechanism. Thus, in rar1, lid cycling is locked into a conformation favoring NB-LRR client degradation, likely via SGT1 and the proteasome. innate immunity | Pseudomonas syringae | SGT1 | STAND ATPase protein

The plant hormone abscisic acid (ABA) acts as a developmental signal and as an integrator of environmental cues such as drought and cold. Key players in ABA signal transduction include the type 2C protein phosphatases (PP2Cs) ABI1 and ABI2, which act by negatively regulating ABA responses. In this study, we identify interactors of ABI1 and ABI2, which we have named regulatory components of ABA receptor (RCARs). In Arabidopsis, RCARs belong to a family with 14 members that share structural similarity with class 10 pathogen-related proteins. RCAR1 was shown to bind ABA, to mediate ABA-dependent inactivation of ABI1 or ABI2 in vitro, and to antagonize PP2C action in planta. Other RCARs also mediated ABA-dependent regulation of ABI1 and ABI2, consistent with a combinatorial assembly of receptor comptexes.

Phytochrome interacting factors (PIFs) are nuclear basic helix-loop-helix (bHLH) transcription factors that negatively regulate photomorphogenesis both in the dark and in the light in Arabidopsis. The phytochrome (phy) family of photoreceptors induces the rapid phosphorylation and degradation of PIFs in response to both red and far-red light conditions to promote photomorphogenesis. Although phys have been shown to function under blue light conditions, the roles of PIFs under blue light have not been investigated in detail. Here we show that PIF1 negatively regulates photomorphogenesis at the seedling stage under blue light conditions, pill seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12 hr light/12 hr dark) blue light conditions. Double-mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cryl, and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants trader diurnal blue light conditions. Blue light induced the rapid phosphorylation, polyubiquitination, and degradation of PIF1 through the ubi/26S proteasomal pathway. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the blue light-induced degradation of PIF1. phyA played a dominant role under pulses of blue light, while phyA, phyB, and phyD induced the degradation of PIF1 in an additive manner under prolonged continuous blue light conditions. Interestingly, the absence of cryl and cry2 enhanced the degradation of PIF1 under blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis under blue light conditions and that blue light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis. DOI: 10.1534/genetics.108.099887

Loss-of-function mutations of SQUINT (SQN)--which encodes the Arabidopsis orthologue of cyclophilin 40 (CyP40)--cause the precocious expression of adult vegetative traits, an increase in carpel number, and produce abnormal spacing of flowers in the inflorescence. Here we show that the vegetative phenotype of sqn is attributable to the elevated expression of miR156-regulated members of the SPL family of transcription factors and provide evidence that this defect is a consequence of a reduction in the activity of ARGONAUTE1 (AGO1). Support for this latter conclusion was provided by the phenotypic similarity between hypomorphic alleles of AG01 and null alleles of SQN and by the genetic interaction between sqn and these alleles. Our results suggest that AGO1, or an AGOl-interacting protein, is a major client of CyP40 and that miR156 and its targets playa central role in the regulation of vegetative phase change in Arabidopsis. CyP40 | HSP90 | miR156 | phase change | immunophilin

SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. This paper presents evidence that the SUMO E3 ligase SIZ1 negatively regulates abscisic acici (ABA) signaling, which is dependent on the bZlP transcription factor ABI5. Loss-of-function T-DNA insertion sizl-2 and sizl-3 mutations caused ABA hypersensitivity for seed germination arrest and seedling primary root growth inhibition. Furthermore, expression of genes that are ABA-responsive through ABI5-dependent signaling (e.g., RD29A, Rd29B, AtEm6, RAB18, ADH1) was hyperinduced by the hormone in sizl seedlings, abi5-4 suppressed ABA hypersensitivity caused by sizl (sizl-2 abi5-4), demonstrating an epistatic genetic interaction between SIZ1 and ABI5. A K391R substitution in ABI5 [ABI5(K391R)] blocked SIZl-mediated sumoylation of the transcription factor in vitro and in Arabidopsis protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. In abi5-4 plants, ABI5(K391R) expression caused greater ABA hypersensitivity (gene expression, seed germination arrest and primary root growth inhibition) compared with ABI5 expression. Together, these results establish that SIZ1dependent sumoylation of ABI5 attenuates ABA signaling. The double mutant sizl-2 afp-1 exhibited even greater ABA sensitivity than the single mutant sizl, suggesting that SIZ1 represses ABI5 signaling function independent of AFP1. abscisic acid | signaling | SUMO | sumoylation

Despite the central importance of stem cells in plant growth and development, the molecular signatures associated with them have not been revealed. Shoot apical meristems (SAMs) harbor a small set of stem cells located at the tip of each plant and they are surrounded by several million differentiating cells. This imposes a major limitation in isolating pure populations of stem cells for genomic analyses. We have developed a system to isolate pure populations of distinct cell types of the SAMs, including stem cells. We have used this system to profile gene expression from 4 different cell samples of SAMs. The cell sample-specific gene expression profiling has resulted in a high-resolution gene expression map to reveal gene expression networks specific to individual spatial domains of SAMs. We demonstrate that the cell sample-specific expression profiling is sensitive in identifying rare transcripts expressed in a few specific subsets of cells of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the spatial expression patterns of genes and it has led to the identification of unique gene expression patterns within the SAMs. Furthermore, our work reveals an enrichment of DNA repair and chromatin modification pathways in stem cells suggesting that maintenance of genome stability and flexible chromatin may be crucial for stem cell function. The gene expression map should guide future reverse genetics experiments, high-resolution analyses of cell-cell communication networks and epigenetic modifications. central zone | CLAVATA3 | fluorescence-activated cell sorting | WUSCHEL

Transcriptional feedback loops constitute the molecular circuitry of the plant circadian dock. In Arabidopsis, a core loop is established between CCA1 and TOC1. Although CCA1 directly represses TOC1, the TOC1 protein has no DNA binding domains, which suggests that it cannot directly regulate CCA1. We established a functional genomic strategy that led to the identification of CHE, a TCP transcription factor that binds specifically to the CCA1 promoter. CHE is a dock component partially redundant with LHY in the repression of CCA1. The expression of CHE is regulated by CCA1, thus adding a CCA1/CHE feedback loop to the Arabidopsis circadian network. Because CHE and TOC1 interact, and CHE binds to the CCA1 promoter, a molecular linkage between TOC1 and CCA1 gene regulation is established.

Information about polymorphism, population structure, and linkage disequilibrium (LD) is crucial for association studies of complex trait variation. However, most genomewide studies have focused on model systems, with very few analyses of undisturbed natural populations. Here, we sequenced 86 mapped nuclear loci for a sample of 46 genotypes of Boechera stricta and two individuals of B. holboellii, both wild relatives of Arabidopsis. Isolation by distance was significant across the species range of B. stricta, and three geographic groups were identified by structure analysis, principal coordinates analysis, and distance-based phylogeny analyses. The allele frequency spectrum indicated a genomewide deviation from an equilibrium neutral model, with silent nucleotide diversity averaging 0.004. LD decayed rapidly, declining to background levels in ~10 kb or less. For tightly linked SNPs separated by

Polyploidy or whole genome duplication (WGD) provides raw genetic materials for sequence and expression evolution of duplicate genes. However, the mode and tempo of expression divergence between WGD duplicate genes in closely related species and recurrent allopolyploids are poorly understood. Arabidopsis is a suitable system for testing the hypothesis that duplicate genes increase expression diversity and regulatory networks. In Arabidopsis, WGD occurred more than once before the split between Arabidopsis thaliana and Arabidopsis arenosa, and both natural and human-made allotetraploids are available. Comparative genomic hybridization analysis indicated that single-copy and duplicate genes after WGD were well preserved in A. thaliana and A. arenosa. Analysis of gene expression microarrays showed that duplicate genes generally had higher levels of expression divergence between two closely related species than single-copy genes. The proportion of the progenitors' duplicate genes that were nonadditively expressed in the resynthesized and natural allotetraploids was significantly higher than that of single-copy genes. Duplicate genes related to environmental stresses tended to be differentially expressed, and multicopy duplicate genes were likely to diverge expression between progenitors and in the allotetraploids. Compared with single-copy genes, duplicate genes tended to contain TATA boxes and less DNA methylation in the promoter regions, facilitating transcriptional regulation by binding transcription factors and/or cis-and trans- acting proteins. The data suggest an important role of WGD duplicate genes in modulating diverse and novel gene expression changes in response to external environmental cues and internal genetic turmoil such as recurrent polyploidy events. evolution | gene expression | polyploidy | stress

Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Downstream effector proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landmark of gene expression control in eukaryotes. Its recognition depends on the presence in the effector protein of a motif termed plant homeodomain (PHD) that specifically binds to H3K4me3. Here, we establish that Arabidopsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. Arabidopsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter region of ORC1 targets. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in Arabidopsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a novel class of H3K4me3 effector proteins characteristic of the plant kingdom. cell cycle transcription | histone H3 lysine 4 methylation | ORC1 DNA replication | plant homeodomain

Two forms of a plant-specific RNA polymerase (Pol), PolIV(PolIVa) and PolV(PolIVb), currently defined by their respective largest subunits [NRPD1(NRPD1a) and NRPE1(NRPD1b)], have been implicated in the production and activity of 24-nt small RNAs (sRNAs) in RNA-directed DNA methylation (RdDM). Prevailing models support the view that PolIV(PolIVa) plays an upstream role in RdDM by producing the 24-nt sRNAs, whereas PolV(PolIVb) would act downstream at a structural rather than an enzymatic level to reinforce sRNA production by PolIV(PolIVa) and mediate DNA methylation. However, the composition and mechanism of action of PolIV-(PolIVa)/PolV(PolIVb) remain unclear. In this work, we have identified a plant-specific PolV(PolIVb) subunit, NRPE5a, homologous to NRPB5a, a common subunit shared by Poll-Ill and shown here to be present in PolIV(PolIVa). Our results confirm the combinatorial diversity of PolIV(PolIVa)/PolV(PolIVb) subunit composition and indicate that these plant-specific Pols are eukaryotic-type polymerases. Moreover, we show that nrpe5a-1 mutation differentially impacts sRNAs accumulation at various PolIV(PolIVa)/PolV(PolIVb)-dependent loci, indicating a target-specific requirement for NRPE5a in the process of PolV(PolIVb)-dependent gene silencing. We then describe that the triad aspartate motif present in the catalytic center of PolV(PolIVb) is required for recapitulation of all activities associated with this Pol complex in RdDM, suggesting that RNA polymerization is important for PolV(PolIVb) to perform its regulatory functions. Arabidopsis | silencing | RNA polymerase

The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-[sup.13][C.sup.2][H.sub.3]]mevalonate with Arabidopsis seedlings. Applying [sup.13]C-{[sup.1]H}{[sup.2]H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol 'the lanosterol pathway.' LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as [beta]-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites. metabolic diversity | mevalonate | sterol | tracer experiment | triterpene

Gene annotation underpins genome science. Most often protein coding sequence is inferred from the genome based on transcript evidence and computational predictions. While generally correct, gene models suffer from errors in reading frame, exon border definition, and exon identification. To ascertain the error rate of Arabidopsis thaliana gene models, we isolated proteins from a sample of Arabidopsis tissues and determined the amino acid sequences of 144,079 distinct peptides by tandem mass spectrometry. The peptides corresponded to 1 or more of 3 different translations of the genome: a 6-frame translation, an exon splice-graph, and the currently annotated proteome. The majority of the peptides (126,055) resided in existing gene models (12,769 confirmed proteins), comprising 40% of annotated genes. Surprisingly, 18,024 novel peptides were found that do not correspond to annotated genes. Using the gene finding program AUGUSTUS and 5,426 novel peptides that occurred in clusters, we discovered 778 new protein-coding genes and refined the annotation of an additional 695 gene models. The remaining 13,449 novel peptides provide high quality annotation (>99% correct) for thousands of additional genes. Our observation that 18,024 of 144,079 peptides did not match current gene models suggests that 13% of the Arabidopsis proteome was incomplete due to approximately equal numbers of missing and incorrect gene models. annotation | genomics | proteomics

Flowering plants have evolved multigene families of the class XI myosin motors, the functions of which remain poorly understood. Here, we investigated functional profiles of the Arabidopsis myosins that belong to two paralogous pairs, XI-K/XI-1 and XI-2/XI-B, using single and double gene-knockout mutants. It was found that the myosins XI-K, XI-2, and XI-B, but not XI-1 have overlapping and additive roles in the root hair elongation. A nonidentical set of the three myosins, XI-K, XI-1, and XI-2, exhibited partially redundant and additive roles in the transport of Golgi stacks, peroxisomes, and mitochondria. Conspicuously, the double xi-k/1 knockout plants that showed the largest cumulative reduction of the organelle velocities also exhibited a stunted plant growth and reduced fecundity phenotype. Collectively, these results suggest that the rapid, myosin-powered organelle trafficking is required for the optimal plant growth, whereas a distinct myosin function, presumably the vesicular transport, is involved in elongation of the root hairs. In addition, our data imply that the myosin gene duplication in plants has been followed by a gradual functional specialization of the resulting pairs of myosin paralogs. myosin | organelle transport | plant development

Vernalization, the acceleration of flowering by winter, involves cold-induced epigenetic silencing of Arabidopsis FLC. This process has been shown to require conserved Polycomb Repressive Complex 2 (PRC2) components including the Su(z)12 homologue, VRN2, and two plant homeodomain (PHD) finger proteins, VRN5 and VIN3. However, the sequence of events leading to FLC repression was unclear. Here we show that, contrary to expectations, VRN2 associates throughout the FLC locus independently of cold. The vernalization-induced silencing is triggered by the cold-dependent association of the PHD finger protein VRN5 to a specific domain in FLC intron 1, and this association is dependent on the cold-induced PHD protein VIN3. In plants returned to warm conditions, VRN5 distribution changes, and it associates more broadly over FLC, coincident with significant increases in H3K27me3. Biochemical purification of a VRN5 complex showed that during prolonged cold a PHD-PRC2 complex forms composed of core PRC2 components (VRN2, SWINGER [an E(Z) HMTase homologue], FIE [an ESC homologue], MSI1 [p55 homologue]), and three related PHD finger proteins, VRN5, VIN3, and VEL1. The PHD-PRC2 activity increases H3K27me3 throughout the locus to levels sufficient for stable silencing. Arabidopsis PHD-PRC2 thus seems to act similarly to Pcl-PRC2 of Drosophila and PHF1-PRC2 of mammals. These data show FLC silencing involves changed composition and dynamic redistribution of Polycomb complexes at different stages of the vernalization process, a mechanism with greater parallels to Polycomb silencing of certain mammalian loci than the classic Drosophila Polycomb targets. Arabidopsis | PHD protein | PRC2 | chromatin modifications

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms (1-6). Recent studies suggest that the methylation status of a number of genes is dynamically [...]

Lateral organ polarity in Arabidopsis is regulated by antagonistic interactions between genes that promote either adaxial or abaxial identity, but the molecular basis of this interaction is largely unknown. We show that the adaxial regulator ASYMMETRIC LEAVES2 (AS2) is a direct target of the abaxial regulator KANADI1 (KAN1), and that KAN1 represses the transcription of AS2 in abaxial cells. Mutation of a single nucleotide in a KAN1 binding site in the AS2 promoter causes AS2 to be ectopically expressed in abaxial cells, resulting in a dominant, adaxialized phenotype. We also show that the abaxial expression of KAN1 is mediated directly or indirectly by AS2. These results demonstrate that KAN1 acts as a transcriptional repressor and that mutually repressive interactions between KAN1 and AS2 contribute to the establishment of adaxial-abaxial polarity in plants.

We survey the distribution of haplotypes at the self-incompatibility (SI) locus of Arabidopsis lyrata (Brassicaceae) at 12 locations spread over the species' natural distribution in Iceland. Previous investigations of the system have identified 34 functionally different S haplotypes maintained by frequency-dependent selection and arranged them into four classes of dominance in their phenotypic expression. On the basis of this model of dominance and the island model of population subdivision, we compare the distribution of S haplotypes with that expected from population genetic theory. We observe 18 different S haplotypes, recessive haplotypes being more common than dominant ones, and dominant ones being shared by fewer populations. As expected, differentiation, although significant, is very low at the S locus even over distances of up to 300 km. The frequency of the most recessive haplotype is slightly larger than expected for a panmictic population, but consistent with a subdivided population with the observed differentiation. Frequencies in nature reflect effects of segregation distortion previously observed in controlled crosses. The dynamics of the S-locus variation are, however, well represented by a 12-island model and our simplified model of dominance interactions.

The CLAVATA1 (CLV1) receptor kinase regulates stem cell specification at shoot and flower meristems of Arabidopsis. Most clv1 alleles are dominant negative, and clv1 null alleles are weak in phenotype, suggesting additional receptors functioning in parallel. We have identified two such parallel receptors, BAM1 and BAM2. We show that the weak nature of the phenotype of clv1 null alleles is dependent on BAM activity, with barn clv mutants exhibiting severe defects in stem cell specification. Furthermore, BAM activity in the meristem depends on CLV2, which is required in part for CLV1 function. In addition, clv1 mutants enhance many of the Barn organ phenotypes, indicating that, contrary to current understanding, CLV1 function is not specific to the meristem. CLV3 encodes a small, secreted peptide that acts as the ligand for CLV1. Mutations in clv3 lead to increased stem cell accumulation. Surprisingly, bam1 and bam2 mutants suppress the phenotype of clv3 mutants. We speculate that in addition to redundant function in the meristem center, BAM1 and BAM2 act to sequester CLV3-like ligands in the meristem flanks.

The plastid plays a vital role in various cellular activities within plant cells including photosynthesis and other metabolic pathways. It is believed that the functional status of the plastid is somehow monitored by the nucleus to optimize the expression of genes encoding plastid proteins. The currently dominant model for plastid-derived signaling ('plastid signaling') proposes that Mg-protoporphyrin IX (MgProto) is a negative signal that represses the expression of a wide range of nuclear genes encoding plastid-localized proteins when plastid development is inhibited. In this study, we have re-evaluated this hypothesis by quantifying the steady-state levels of MgProto (as well as its neighboring intermediates protoporphyrin IX and MgProto monomethyl ester [MgProtoMe]) in Arabidopsis plants with altered plastid signaling responses as monitored by expression of the Lhcb1, RBCS, HEMA1, BAM3 and CA1 genes. In addition, we have examined the correlation between gene expression and MgProto (MgProtoMe) in a range of mutants and conditions in which the steady-state levels of MgProto (MgProtoMe) have been modified. Overall we found that there was no correlation between the steady-state levels of MgProto (MgProtoMe) and Lhcb1 expression or with any of the other genes tested. Taking these results together, we propose that the current model on plastid signaling must be revised. gene expression | retrograde signal | tetrapyrrole

Chloroplast biogenesis involves careful coordination of both plastid and nuclear gene expression, which is achieved in part by retrograde signaling from the chloroplast to the nucleus. This can be demonstrated by the fact that the herbicide, Norflurazon (NF), which causes bleaching of chloroplasts, prevents the light induction of photosynthesis-related genes in the nucleus. It has been proposed that the tetrapyrrole pathway intermediate Mg-protoporphyrin IX acts as the signaling molecule in this pathway and accumulates in the chloroplasts and cytosol of the cell after NF treatment. Here we present data that demonstrate that this model is too simplistic. We have developed a sensitive liquid chromatography-mass spectrometry (LC/MS) method to measure tetrapyrrole intermediates and have shown that no Mg-protoporphyrin IX, nor indeed any other chlorophyll-biosynthesis intermediate, can be detected in NF-treated plants under conditions in which nuclear gene expression is repressed. Conversely when endogenous Mg-protoporphyrin IX levels are artificially increased by supplementation with the tetrapyrrole precursor, 5-aminolevulinic acid, the expression of nuclear-encoded photosynthetic genes is induced, not repressed. We also demonstrate that NF-treatment leads to a strong down-regulation of tetrapyrrole biosynthesis genes, consistent with the absence of an accumulation of tetrapyrrole intermediates. Finally, there is no correlation between nuclear-gene expression and any of the chlorophyll biosynthetic intermediates over a range of growth conditions and treatments. Instead, it is possible that a perturbation of tetrapyrrole synthesis may lead to localized ROS production or an altered redox state of the plastid, which could mediate retrograde signaling.

Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis. interpathway regulation | plant antiviral defense

Transposable elements (TEs) are the major component of most plant genomes, and characterizing their population dynamics is key to understanding plant genome complexity. Yet there have been few studies of TE population genetics in plant systems. To study the roles of selection, transposition, and demography in shaping TE population diversity, we generated a polymorphism dataset for six TE families in four populations of the flowering plant Arabidopsis lyrata. The TE data indicated significant differentiation among populations, and maximum likelihood procedures suggested weak selection. For strongly bottlenecked populations, the observed TE band-frequency spectra fit data simulated under neutral demographic models constructed from nucleotide polymorphism data. Overall, we propose that TEs are subjected to weak selection, the efficacy of which varies as a function of demographic factors. Thus, demographic effects could be a major factor driving distributions of TEs among plant lineages. bottleneck | genetics | TE-display