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Post-transcriptional regulation makes an important contribution to adjusting the transcriptome to environmental changes in plants. RNA-binding proteins are key players that interact specifically with mRNAs to co-ordinate their fate. While the regulatory interactions between proteins and RNA are well understood in animals, until recently little information was available on the global binding landscape of RNA-binding proteins in higher plants. This is not least due to technical challenges in plants. In turn, while numerous RNA-binding proteins have been identified through mutant analysis and homology-based searches in plants, only recently a full compendium of proteins with RNA-binding activity has been experimentally determined for the reference plant Arabidopsis thaliana. State-of-the-art techniques to determine RNA-protein interactions genome-wide in animals are based on the covalent fixation of RNA and protein in vivo by UV light. This has only recently been successfully applied to plants. Here, we present practical considerations on the application of UV irradiation based methods to comprehensively determine in vivo RNA-protein interactions in Arabidopsis thaliana, focussing on individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) and mRNA interactome capture. Copyright © 2019 Elsevier Inc. All rights reserved.

BackgroundAccurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single molecule long read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation or incomplete cDNA synthesis.ResultsWe present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 160k transcripts - twice that of the best current Arabidopsis transcriptome and including over 1,500 novel genes. 79% of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We developed novel methods to determine splice junctions and transcription start and end sites accurately. Mis- match profiles around splice junctions provided a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identified high confidence transcription start/end sites and removed fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provided higher resolution of transcript expression profiling and identified cold- and light-induced differential transcription start and polyadenylation site usage.ConclusionsAtRTD3 is the most comprehensive Arabidopsis transcriptome currently available. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single molecule sequencing analysis from any species.

The timing of floral initiation is a tightly controlled process in plants. The circadian clock regulated glycine-rich RNA-binding protein AtGRP7, a known regulator of splicing, was previously shown to regulate flowering time mainly by affecting the MADS-box repressor FLC. Loss of AtGRP7 leads to elevated FLC expression and late flowering in the atgrp7-1 mutant. Here, we analyze genetic interactions of AtGRP7 with key regulators of the autonomous and the thermosensory pathway of floral induction. RNAi mediated reduction of the level of the paralogous AtGRP8 in atgrp7-1 further delays floral transition compared to atgrp7-1. AtGRP7 acts in parallel to FCA, FPA and FLK in the branch of the autonomous pathway comprised of RNA-binding proteins. It acts in the same branch as FLD, and AtGRP7 loss-of-function mutants show elevated levels of di-methylated lysine 4 of histone H3, a mark for active transcription. In addition to its role in the autonomous pathway, AtGRP7 acts in the thermosensory pathway of flowering time control by regulating alternative splicing of the floral repressor FLM. Overexpression of AtGRP7 selectively favors formation of the repressive isoform FLM-beta. Our results suggest that the RNA-binding proteins AtGRP7 and AtGRP8 influence MADS-Box transcription factors in at least two different pathways of flowering time control. This highlights the importance of RNA-binding proteins to fine-tune the integration of varying cues into flowering time control and further strengthens the view that the different pathways, although genetically separable, constitute a tightly interwoven network to ensure plant reproductive success under changing environmental conditions. © The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins., eLife digest Genes are strings of genetic code that contain instructions for producing a cell’s proteins. Active genes are copied from DNA into molecules called mRNAs, and mRNA molecules are subsequently translated to create new proteins. However, the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA. Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production. One of these methods involves adding a chemical tag, a methyl group, onto mRNA while it is being created. These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation. Most eukaryotic species – which include animals, plants and fungi – use the same system to add methyl tags to mRNA molecules. One methyl tag in particular, known as m6A, is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants. However, in the flowering plant Arabidopsis thaliana, ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag, creating obvious confusion about how the system works in plants. Arribas-Hernández, Rennie et al. investigated this question using advanced large-scale biochemical techniques, and discovered that conventional m6A methyl tags are indeed used by ECT2 in Arabidopsis thaliana. The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m6A tags, possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA. Arribas-Hernández, Rennie et al. also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding. These findings provide new insights into how chemical labels in mRNA control gene activity. They have broad implications that extend beyond plants into other eukaryotic species, including humans. Since this chemical labelling system has a major role in controlling plant growth, these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production.

The receptor protein kinase FER regulates plant growth and stress responses through alternative splicing., The environmentally responsive signaling pathways that link global transcriptomic changes through alternative splicing (AS) to plant fitness remain unclear. Here, we found that the interaction of the extracellular rapid alkalinization FACTOR 1 (RALF1) peptide with its receptor FERONIA (FER) triggered a rapid and massive RNA AS response by interacting with and phosphorylating glycine-rich RNA binding protein7 (GRP7) to elevate GRP7 nuclear accumulation in Arabidopsis thaliana. FER-dependent GRP7 phosphorylation enhanced its mRNA binding ability and its association with the spliceosome component U1-70K to enable splice site selection, modulating dynamic AS. Genetic reversal of a RALF1-FER–dependent splicing target partly rescued mutants deficient in GRP7. AS of GRP7 itself induced nonsense-mediated decay feedback to the RALF1-FER-GRP7 module, fine-tuning stress responses, and cell growth. The RALF1-FER-GRP7 module provides a paradigm for regulatory mechanisms of RNA splicing in response to external stimuli.

In recent years, it has become increasingly recognized that regulation at the RNA level pervasively shapes the transcriptome in eukaryotic cells. This has fostered an interest in the mode of action of RNA-binding proteins that, via interaction with specific RNA sequence motifs, modulate gene expression. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In metazoans and yeast, methods have been devised to stabilize RNA-protein interactions by UV cross-linking before isolating RNA-protein complexes using antibodies, followed by identification of associated RNAs by next-generation sequencing. These methods are collectively referred to as CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing). Here, we present a version of the individual nucleotide resolution cross-linking and immunoprecipitation procedure that is suitable for use in the model plant Arabidopsis thaliana.

N6-methyladenosine (m6A) emerges as an important modification in eukaryotic mRNAs. m6A has first been reported in 1974, and its functional significance in mammalian gene regulation and importance for proper development has been well established. An arsenal of writer, eraser, and reader proteins accomplish deposition, removal, and interpretation of the m6A mark, resulting in dynamic function. This led to the concept of an epitranscriptome, the compendium of RNA species with chemical modification of the nucleobases in the cell, in analogy to the epigenome. While m6A has long been known to also exist in plant mRNAs, proteins involved in m6A metabolism have only recently been detected by mutant analysis, homology search, and mRNA interactome capture in the reference plant Arabidopsis thaliana. Dysregulation of the m6A modification causes severe developmental abnormalities of leaves and roots and altered timing of reproductive development. Furthermore, m6A modification affects viral infection. Here, we discuss recent progress in identifying m6A sites transcriptome-wide, in identifying the molecular players involved in writing, removing, and reading the mark, and in assigning functions to this RNA modification in Arabidopsis thaliana. We highlight similarities and differences to m6A modification in mammals and provide an outlook on important questions that remain to be addressed. © The Author(s) 2019. Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

A distinct chromatin mark, H3K36me3, has been found to engage in temperature-dependent alternative splicing and ambient temperature-dependent flowering-time control in Arabidopsis.

Alternative splicing is a molecular tool of the cell to generate more than one messenger RNA from the same gene. Through variable combinations of exons blueprints for different proteins are assembled from one and the same pre-messenger RNA, thus increasing the complexity of the proteome. Moreover, through alternative splicing different transcript variants with different stabilities and different regulatory motifs can be generated, leading to variation in the transcriptome. The importance of alternative splicing in plants has been increasingly recognized in the last decade. Alternative splicing has been found during abiotic and biotic stress and during development. Here, recent advancements in the understanding of alternative splicing in higher plants are presented. Mechanistic details and functional consequences of alternative splicing are discussed with a focus on the model plant Arabidopsis thaliana.

Background Functions for RNA-binding proteins in orchestrating plant development and environmental responses are well established. However, the lack of a genome-wide view of their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) genome-wide to determine the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7. Results iCLIP identifies 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP that are absent in plants expressing an RNA-binding-dead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. AtGRP7 can bind to all transcript regions, with a preference for 3′ untranslated regions. In the vicinity of crosslink sites, U/C-rich motifs are overrepresented. Cross-referencing the targets against transcriptome changes in AtGRP7 loss-of-function mutants or AtGRP7-overexpressing plants reveals a predominantly negative effect of AtGRP7 on its targets. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels. Conclusions We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1332-x) contains supplementary material, which is available to authorized users.

Plants precisely time the onset of flowering to ensure reproductive success. A major factor in seasonal control of flowering time is the photoperiod. The length of the daily light period is measured by the circadian clock in leaves, and a signal is conveyed to the shoot apex to initiate floral transition accordingly. In the last two decades, the molecular players in the photoperiodic pathway have been identified in Arabidopsis thaliana. Moreover, the intricate connections between the circadian clockwork and components of the photoperiodic pathway have been unravelled. In particular, the molecular basis of time-of-day-dependent sensitivity to floral stimuli, as predicted by Bünning and Pittendrigh, has been elucidated. This review covers recent insights into the molecular mechanisms underlying clock regulation of photoperiodic responses and the integration of the photoperiodic pathway into the flowering time network in Arabidopsis. Furthermore, examples of conservation and divergence in photoperiodic flower induction in other plant species are discussed.

Plants show a suite of inducible defense responses against bacterial pathogens. Here we investigate in detail the effect of the circadian clock on these reactions in Arabidopsis thaliana. The magnitude of immune responses elicited by flg22, by virulent and by avirulent Pseudomonas syringae strains depends on the time of day of inoculation. The oxidative burst is stronger when flg22 is infiltrated in the morning in wild-type plants but not in the arrhythmic clock mutant lux arrhythmo/phytoclock1 (pcl1), and thus is controlled by the endogenous clock. Similarly, when bacteria are syringe-infiltrated into the leaf, defense gene induction is higher and bacterial growth is suppressed more strongly after morning inoculation in wild-type but not in pcl1 plants. Furthermore, cell death associated with the hypersensitive response was found to be under clock control. Notably, the clock effect depends on the mode of infection: upon spray inoculation onto the leaf surface, defense gene induction is higher and bacterial growth is suppressed more strongly upon evening inoculation. This different phasing of pre-invasive and post-invasive defense relates to clock-regulated stomatal movement. In particular, TIME FOR COFFEE may impact pathogen defense via clock-regulated stomata movement apart from its known role in time-of-day-dependent jasmonate responses. Taken together, these data highlight the importance of the circadian clock for the control of different immune responses at distinct times of the day. The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

RNA-protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture - where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

This study focused on the identification and phylogenetic analysis of glycine-rich RNA binding proteins that contain an RNA recognition motif (RRM)-type RNA binding domain in addition to a region with contiguous glycine residues in representative plant species. In higher plants, glycine-rich proteins with an RRM have met considerable interest as they are responsive to environmental cues and play a role in cold tolerance, pathogen defense, flowering time control, and circadian timekeeping. To identify such RRM containing proteins in plant genomes we developed an RRM profile based on the known glycine-rich RRM containing proteins in the reference plant Arabidopsis thaliana. The application of this remodeled RRM profile that omitted sequences from non-plant species reduced the noise when searching plant genomes for RRM proteins compared to a search performed with the known RRM_1 profile. Furthermore, we developed an island scoring function to identify regions with contiguous glycine residues, using a sliding window approach. This approach tags regions in a protein sequence with a high content of the same amino acid, and repetitive structures score higher. This definition of repetitive structures in a fixed sequence length provided a new glance for characterizing patterns which cannot be easily described as regular expressions. By combining the profile-based domain search for well-conserved regions (the RRM) with a scoring technique for regions with repetitive residues we identified groups of proteins related to the A. thaliana glycine-rich RNA binding proteins in eight plant species.

The phenological development of plants can be broadly divided into 4 stages, embryo/juvenile, adult (all vegetative stages), reproductive (the generative stage), and senescent. This research topic focusses on the transition from vegetative growth to reproductive development, commonly referred to as floral transition. Plants have coordinated the seasonal timing of flowering and reproduction with the prevailing environmental conditions. In agriculture, flowering is a prerequisite for crop production whenever seeds or fruits are harvested. In contrast, avoidance of flowering is necessary for harvesting vegetative parts of a plant such as tubers or roots. Late flowering also severely hampers breeding success due to long generation times. Thus, flowering time regulation is of utmost importance for genetic improvement of crops.

Alternative splicing (AS) of pre-mRNAs is an important regulatory mechanism shaping the transcriptome. In plants, only few RNA-binding proteins are known to affect AS. Here, we show that the glycine-rich RNA-binding protein AtGRP7 influences AS in Arabidopsis thaliana. Using a high-resolution RT-PCR-based AS panel, we found significant changes in the ratios of AS isoforms for 59 of 288 analyzed AS events upon ectopic AtGRP7 expression. In particular, AtGRP7 affected the choice of alternative 5' splice sites preferentially. About half of the events are also influenced by the paralog AtGRP8, indicating that AtGRP7 and AtGRP8 share a network of downstream targets. For 10 events, the AS patterns were altered in opposite directions in plants with elevated AtGRP7 level or lacking AtGRP7. Importantly, RNA immunoprecipitation from plant extracts showed that several transcripts are bound by AtGRP7 in vivo and indeed represent direct targets. Furthermore, the effect of AtGRP7 on these AS events was abrogated by mutation of a single arginine that is required for its RNA-binding activity. This indicates that AtGRP7 impacts AS of these transcripts via direct interaction. As several of the AS events are also controlled by other splicing regulators, our data begin to provide insights into an AS network in Arabidopsis.

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.7-Å resolution reveals two unique protruding loops, L1 and L4, not found in other mono-ADP-ribosyltransferases. Site-directed mutagenesis demonstrates that these loops are essential for substrate recognition and enzymatic activity. HopU1 ADP-ribosylates the conserved arginine 49 of GRP7, and this reduces the ability of GRP7 to bind RNA in vitro. In vivo, expression of GRP7 with Arg-49 replaced with lysine does not complement the reduced immune responses of the Arabidopsis thaliana grp7-1 mutant demonstrating the importance of this residue for GRP7 function. These data provide mechanistic details how HopU1 recognizes this novel type of substrate and highlights the role of GRP7 in plant immunity.

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post-transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5' splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5' half of the intron with a premature termination codon. In mutants defective in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indicating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

The RNA binding protein AtGRP7 is part of a circadian slave oscillator in Arabidopsis thaliana that negatively autoregulates its own mRNA, and affects the levels of other transcripts. Here, we identify a novel role for AtGRP7 as a flowering-time gene. An atgrp7-1 T-DNA mutant flowers later than wild-type plants under both long and short days, and independent RNA interference lines with reduced levels of AtGRP7, and the closely related AtGRP8 protein, are also late flowering, particularly in short photoperiods. Consistent with the retention of a photoperiodic response, the transcript encoding the key photoperiodic regulator CONSTANS oscillates with a similar pattern in atgrp7-1 and wild-type plants. In both the RNAi lines and in the atgrp7-1 mutant transcript levels for the floral repressor FLC are elevated. Conversely, in transgenic plants ectopically overexpressing AtGRP7, the transition to flowering is accelerated mainly in short days, with a concomitant reduction in FLC abundance. The late-flowering phenotype of the RNAi lines is suppressed by introducing the flc-3 loss-of-function mutation, suggesting that AtGRP7 promotes floral transition, at least partly by downregulating FLC. Furthermore, vernalization overrides the late-flowering phenotype. Retention of both the photoperiodic response and vernalization response are features of autonomous pathway mutants, suggesting that AtGRP7 is a novel member of the autonomous pathway.

Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.

Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

The circadian clock serves to prepare plants for predictable changes in the environment both on a daily and a seasonal basis. Floral transition in most plants is dependent on day length and, thus, on the season. Sensing day length allows plants to synchronize their reproduction with favorable outside conditions. This process is known as the photoperiodic pathway of flowering time control, and it is tightly linked with the circadian clock of the plant. Thus, mutants with defects in the circadian system can also be impaired in photoperiodic flower induction. This chapter provides a detailed description of the methods used to determine flowering time in the model species Arabidopsis thaliana and Hordeum vulgare and to assign a mutant or a transgenic plant to the photoperiodic pathway of flowering.

Summary SRR1 regulates flowering time in Arabidopsis by integrating photoperiodic and photoperiod-independent signals. By promoting expression of several repressors of FT, SRR1 represses flowering in non-inductive conditions., Timing of flowering is determined by environmental and developmental signals, leading to promotion or repression of key floral integrators. SENSITIVITY TO RED LIGHT REDUCED (SRR1) is a pioneer protein previously shown to be involved in regulation of the circadian clock and phytochrome B signalling in Arabidopsis thaliana. This report has examined the role of SRR1 in flowering time control. Loss-of-function srr1-1 plants flowered very early compared with the wild type under short-day conditions and had a weak flowering response to increasing daylength. Furthermore, FLOWERING LOCUS T (FT) transcript levels were elevated already in short days in srr1-1 compared with the wild type. This correlated with elevated end of day levels of CONSTANS (CO), whereas levels of CYCLING DOF FACTOR 1 (CDF1), a repressor of CO transcription, were reduced. srr1-1 gi-2 and srr1-1 co-9 double mutants showed that SRR1 can also repress flowering independently of the photoperiodic pathway. srr1-1 flowered consistently early between 16 °C and 27 °C, showing that SRR1 prevents premature flowering over a wide temperature range. SRR1 also promotes expression of the repressors TEMPRANILLO 1 (TEM1) and TEM2. Consequently their targets in the gibberellin biosynthesis pathway were elevated in srr1-1. SRR1 is thus an important focal point of both photoperiodic and photoperiod-independent regulation of flowering. By stimulating expression of the FT-binding repressors CDF1, TEM1 and TEM2, and FLC, flowering is inhibited in non-inductive conditions.

The RNA-binding protein Arabidopsis thaliana glycine-rich RNA-binding protein 7 (AtGRP7) regulates the steady-state abundance of numerous target transcripts in A. thaliana. Here we show that the GA1 and GA2 transcripts encoding the first enzymes of the gibberellin biosynthetic pathway are expressed at reduced levels in transgenic plants ectopically over-expressing AtGRP7 (AtGRP7-ox plants). Furthermore, the levels of the bioactive phytohormone GA(4) as well as of several intermediates of the GA biosynthetic pathway are reduced in AtGRP7-ox plants. The transgenic plants show a reduced length of the vegetative stem. The application of exogenous GA largely reverses the phenotype by increasing the number of vegetative internodes. AtGRP7-ox plants flower with fewer leaves than wt plants, suggesting that the floral promotive effect of AtGRP7 bypasses the effect of a reduced GA level in AtGRP7-ox plants. Upon GA treatment, AtGRP7-ox plants flower only slightly earlier than wild type plants. Thus, exogenous GA has only a small additional effect in reducing the number of leaves at the onset of flowering in AtGRP7-ox plants.

AnArabidopsistranscript preferentially expressed at the end of the daily light period codes for the RNA–binding proteinAtGRP7. A reverse genetic approach inArabidopsis thalianahas revealed its role in the generation of circadian rhythmicity:AtGRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit:Atgrp7 gene transcription is rhythmically activated by the circadian clock during the day. TheAtGPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post–transcriptional level. In this respect, theAtGRP7 feedback loop differs from known circadian oscillators in the fruitflyDrosophilaand mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that theAtGRP7 feedback loop may act within an output pathway from theArabidopsisclock.

Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germin's unusual biochemical properties and oxalate oxidase activity. Using specific antibodies, we have studied three GLPs (AtGER1, AtGER2 and AtGER3) in Arabidopsis thaliana (L.) Heynh. as well as in transgenic tobacco ( Nicotiana tabacum L.) plants overexpressing these proteins. Like wheat ( Triticum aestivum L.) germin, these Arabidopsis GLPs are associated with the extracellular matrix (ECM) and they also seem to exist as two glycosylated isoforms. However, none of them is an oxalate oxidase. Although GLPs display several conserved features, each has its specific characteristics. Both AtGER2 and AtGER3 are oligomeric proteins that share germin's resistance to pepsin and to dissociation by heat and SDS. In contrast, AtGER1 seems to exist as a monomer. The GLPs may interact with the ECM in a variety of ways, since each is efficiently extracted by different conditions. In addition, germins and GLPs all bind Cibacron Blue, a dye often but not exclusively used for the purification of enzymes having nucleotide cofactors. In the case of AtGER2, binding to the dye is so tight that it almost allows a one-step purification of this protein. The variety of sequences, expression patterns and biochemical features indicates that GLPs could be a class of receptors localized in the ECM and involved in physiological and developmental processes as well as stress response.

In Arabidopsis thaliana, steady-state abundance of the Atger3 transcript encoding a germin-like cell wall protein follows a circadian rhythm, reaching its highest level at the beginning of the night. As a first step towards dissecting the molecular mechanisms underlying these transcript oscillations, the Atger3 genomic locus was characterised. Transcriptional fusions of 1.8 kb and 967 bp Atger3 promoter fragments to the β-glucuronidase (GUS) reporter gene mediate high-amplitude circadian oscillations of the GUS transcript in transgenic Arabidopsis. 5′ deletion to −490 greatly reduces overall transcript abundance while retaining a basal oscillation. Further deletion to −299 abolishes preferential GUS expression in the evening. Taken together, these data indicate that clock-response elements contributing to high-amplitude Atger3 oscillations largely reside between −299 and −967. Histochemical staining for GUS activity indicates that the Atger3 promoter is active in cotyledons, young leaves, petioles, the inflorescence axis, pedicels, sepals, ovary, style and siliques but not in roots, petals and anthers.

The endogenous clock that drives circadian rhythms is thought to communicate temporal information within the cell via cycling downstream transcripts. A transcript encoding a glycine-rich RNA-binding protein, Atgrp7 , in Arabidopsis thaliana undergoes circadian oscillations with peak levels in the evening. The At GRP7 protein also cycles with a time delay so that Atgrp7 transcript levels decline when the At GRP7 protein accumulates to high levels. After At GRP7 protein concentration has fallen to trough levels, Atgrp7 transcript starts to reaccumulate. Overexpression of At GRP7 in transgenic Arabidopsis plants severely depresses cycling of the endogenous Atgrp7 transcript. These data establish both transcript and protein as components of a negative feedback circuit capable of generating a stable oscillation. At GRP7 overexpression also depresses the oscillation of the circadian-regulated transcript encoding the related RNA-binding protein At GRP8 but does not affect the oscillation of transcripts such as cab or catalase mRNAs. We propose that the At GRP7 autoregulatory loop represents a “slave” oscillator in Arabidopsis that receives temporal information from a central “master” oscillator, conserves the rhythmicity by negative feedback, and transduces it to the output pathway by regulating a subset of clock-controlled transcripts.

Wheat germin is a protein expressed during germination which possesses an oxalate oxidase activity. Germin-type oxalate oxidases have been extensively studied in monocotyledons (wheat and barley) where they are thought to have important functions for development, stress response and defence against pathogens. In contrast, almost nothing is known about the germin-like proteins found in dicotyledons, gymnosperms and myxomycetes. In this work, cDNA clones for three genes (ATGER1, ATGER2 and ATGER3) encoding germin-like proteins, initially characterized as expressed sequence tags (ESTs), from Arabidopsis thaliana cDNA libraries were further characterized. In addition, we isolated and sequenced a Brassica napus cDNA which was strongly homologous to the cDNA for ATGER1. Sequence analysis and secondary structure predictions of the proteins encoded by these cDNAs showed that they possess all the characteristic features of members of the germin family and of the germin/seed globulins/sucrose binding protein superfamily. Sequence comparisons and mapping demonstrated the existence of at least two different gene families in the A. thaliana genome encoding a minimum of three genes for germins. These three genes have been mapped in three different location on the Arabidopsis genome. By northern blot hybridizations we found that these genes are differentially regulated. ATGER1 was expressed during germination, like wheat germin, but also in leaves whereas ATGER2 transcripts were exclusively found in developing embryos, like wheat pseudo-germin. ATGER3 mRNAs were found in leaves and flowers and their abundance was shown to vary during the circadian cycle.

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the 'positively switchable' PADRON C-s with the 'negatively switchable' rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.

Background: In angiosperms, RNA-binding proteins with an RNA recognition motif (RRM)-type RNA interaction domain play an important role in developmental and environmental responses. Despite their pivotal role, a comprehensive analysis of their number and diversity has only been performed in Arabidopsis so far. Results: Here we present a detailed phylogenetic analysis of RRM-containing proteins in plants, the red algae Cyanidioschyzon merolae and cyanobacteria. We identified two major events during the diversification of the RRM in plants, one at the emergence of green plants, and the other at the water-to-land transition. We focused on proteins that combine a single RRM with a glycine-rich stretch, known as glycine-rich RNA-binding proteins (GRPs). We found that GRPs are present in cyanobacteria, however plant and cyanobacterial GRPs are not of monophyletic origin. We provide evidence that plant GRPs form a polyphyletic group.*Conclusion:*Our work provides insights into the origin of GRPs in plants. We determined that the RRM from plants and cyanobacteria do not have a common origin. We could also determine that the acquisition of the glycine-rich stretch has happened at least on three separate occasions during the evolution of GRPs. One event led to the emergence of cyanobacterial GRPs, while later acquisition events led to the emergence of GRPs in the green lineage. No GRPs were found in red or marine green algae. We found a subgroup of GRPs exclusive to land plants, and its appearance may be linked to challenges related to the water-to-land transition.

The circadian clock is an endogenous, approximately 24-h timer that enables plants to anticipate daily changes in their environment and regulates a considerable fraction of the transcriptome. At the core of the circadian system is the oscillator, made up of interconnected feedback loops, involving transcriptional regulation of clock genes and post-translational modification of clock proteins. Recently, it has become clear that post-transcriptional events are also critical for shaping rhythmic mRNA and protein profiles. This review covers regulation at the RNA level of both the core clock and output genes in Arabidopsis (Arabidopsis thaliana), with comparisons with other model organisms. We discuss the role of splicing, mRNA decay and translational regulation as well as recent insights into rhythms of noncoding regulatory RNAs.

The protein Slr0782 from Synechocystis sp. PCC 6803, which has similarity to L-amino acid oxidase from Synechococcus elongatus PCC 6301 and PCC 7942, has been characterized in part. Immunoblot blot analysis showed that Slr0782 is mainly thylakoid membrane-associated. Moreover, expression of slr0782 mRNA and Slr0782 protein were analyzed and an activity assay was developed. Utilizing toluene-permeabilized cells, an L-arginine-stimulated O(2) uptake became detectable in Synechocystis sp. PCC 6803. Besides oxidizing the basic L-amino acids L-arginine, L-lysine, L-ornithine, and L-histidine, a number of other L-amino acids were also substrates, while D-amino acids were not. The best substrate was L-cysteine, and the second best was L-arginine. The L-arginine-stimulated O(2) uptake was inhibited by cations. The inhibition by o-phenanthroline and salicylhydroxamic acid suggested the presence of a transition metal besides FAD in the enzyme. Moreover, it is shown that inhibitors of the respiratory electron transport chain, such as KCN and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, also inhibited the L-arginine-stimulated O(2) uptake, suggesting that Slr0782 functions as an L-arginine dehydrogenase, mediating electron transfer from L-arginine into the respiratory electron transport chain utilizing O(2) as electron acceptor via cytochrome oxidase. The results imply that Slr0782 is an additional substrate dehydrogenase being able to interact with the electron transport chain of the thylakoid membrane.

Background So far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis. Results We have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i) an L-arginine decarboxylase pathway, (ii) an L-arginine deiminase pathway, and (iii) an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 μmol photons m-2 s-1 showed that the transcripts for the first enzyme(s) of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase. Conclusion The evaluation of 24 cyanobacterial genomes revealed that five different L-arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L-arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways were constitutively expressed with the exception of the transcript for the carbamate kinase, which was substantially up-regulated in cells grown with L-arginine.

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 ( also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7' s ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome.

Although rhythmic leaf movement in a higher plant was the first physiological process recognised to be under circadian control, our understanding of the molecular drives underlying circadian rhythms in plants is still limited. Genetic screens for mutants impaired with regard to circadian rhythmicity have identified components critical for clock function in the model plant Arabidopsis thaliana, providing a snapshot of interconnected transcription-based feedback circuits at the core of the clockwork. Subsequently, the Arabidopsis genome project provided the basis for reverse genetic approaches to uncover additional gene products operating close to the core clockwork. We will review recent progress in the dissection of the molecular mechanisms within the basic oscillator and in the incorporation of additional components into the basic clock model.

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that At TRN1 binds to similar domains in hnRNP-like proteins from plants. At TRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-ß family, At TRN1 binds to the regulatory GTPase Ran from Arabidopsis . We demonstrated that the amino terminus of At TRN1 is necessary for this interaction. Recombinant At TRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with At TRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis , At GRP7 and At GRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled At GRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis At TRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.

Plants possess several photoreceptors to sense the light environment. InArabidopsiscryptochromes and phytochromes play roles in photomorphogenesis and in the light input pathways that synchronize the circadian clock with the external world. We have identifiedSRR1(sensitivity tored lightreduced), a gene that plays an important role in phytochrome B (phyB)-mediated light signaling. The recessivesrr1null allele andphyBmutants display a number of similar phenotypes indicating thatSRR1is required for normal phyB signaling. Genetic analysis suggests thatSRR1works both in the phyB pathway but also independently of phyB.srr1mutants are affected in multiple outputs of the circadian clock in continuous light conditions, including leaf movement and expression of the clock components,CCA1andTOC1. Clock-regulated gene expression is also impaired during day–night cycles and in constant darkness. The circadian phenotypes ofsrr1mutants in all three conditions suggest that SRR1 activity is required for normal oscillator function. TheSRR1gene was identified and shown to code for a protein conserved in numerous eukaryotes including mammals and flies, implicating a conserved role for this protein in both the animal and plant kingdoms.

To keep track of fluctuations in spectral composition and intensity of incoming sunlight, plants engage a plethora of photosensory pigments. Absorption of light by these photoreceptors sets in motion signaling cascades that ultimately influence the plant's physiology. Many light-controlled processes are based on modulation of gene activity in response to changes in irradiation. The molecular basis of this regulation and the downstream components transducing signals from the photoreceptors are not fully understood yet, but recent evidence suggests that some of those routes are rather short. The phytochrome photoreceptors have been found to influence light-responsive promoters by direct contact with transcription factors. Additionally, the cryptochrome blue-light receptors directly interact with a key repressor of photomorphogenesis, suggesting that light activation of photoreceptors could initiate photomorphogenesis through posttranslational regulation. This review focuses on recent insights into photosensory transduction mechanisms as well as on our current understanding of light entrainment of the endogenous clock.

Molecular models have been described for the circadian clocks of representatives of several different taxa. Much of the work on the plant circadian system has been carried out using the thale cress,Arabidopsis thaliana, as a model. We discuss the roles of genes implicated in the plant circadian system, with special emphasis onArabidopsis. Plants have an endogenous clock that regulates many aspects of circadian and photoperiodic behaviour. Despite the discovery of components that resemble those involved in the clocks of animals or fungi, no coherent model of the plant clock has yet been proposed. In this review, we aim to provide an overview of studies of theArabidopsiscircadian system. We shall compare these with results from different taxa and discuss them in the context of what is known about clocks in other organisms.

It is now widely accepted that autoregulatory circuits involving transcription/translation of clock genes form the molecular basis of the endogenous circadian clock in different organisms. In Arabidopsis thaliana, the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich protein) has been identified as part of a negative-feedback loop through which AtGRP7 regulates the circadian oscillations of its own transcript. Experimental evidence indicates that this feedback loop also is influenced by another oscillator. Support for this hypothesis comes from the characterization of the clock mutant toc1 (timing of cab expression) and the recent isolation of two candidate clock molecules, LHY (late elongated hypocotyl) and CCA1 (circadian clock associated). TOC1, as well as the LHY and CCA1 oscillatory feedback loops, influence several rhythmic physiological and molecular processes in Arabidopsis, including cyclic Atgrp7 gene expression. We discuss the features of these feedback loops with relation to the organization of the circadian system in Arabidopsis.

The Atgrp7 transcript encodes a clock-regulated, glycine-rich, RNA-binding protein in Arabidopsis thaliana and shows a circadian variation in steady-state abundance. Constitutive overexpression of its product, At GRP7, in transgenic Arabidopsis plants depresses the oscillations of the endogenous Atgrp7 transcript, indicating that both the transcript and the protein are part of a clock-regulated negative feedback circuit. Here we characterise the upstream region of the Atgrp7 gene in order to begin to dissect the molecular basis of this oscillating autoregulatory feedback loop. Fusion of a 1.5-kb promoter fragment to the β-glucuronidase ( gus ) reporter gene leads to circadian oscillations in the level of the gus transcript in transgenic Arabidopsis plants, with highest levels in the evening, indicating that transcription of the Atgrp7 gene is rhythmically activated by the endogenous circadian clock. A 265-bp fragment upstream of the transcription start site is necessary for high-amplitude Atgrp7 cycling. Within this region, a 56-bp clock-responsive element that confers a low-amplitude circadian oscillation (approximately threefold) with peak abundance in the early evening maps between positions −112 and −57. Another element necessary for augmenting the amplitude of the oscillation lies between −178 and −264. Genetic crosses between a line bearing a promoter- gus fusion and plants that overexpress At GRP7 show that the promoter by itself does not mediate the negative feedback of At GRP7 on the oscillations of its own transcript.

Volume 30 of the Annual Plant Reviews Series, edited by Garry C. Whitelam and Karen J. Halliday, focuses on the impact of light on plant development. The first part sets the stage by providing detailed, state-of-the-art descriptions of the traditional phytochrome, cryptochrome and phototropin photoreceptor families with special emphasis on knowledge gained in Arabidopsis thaliana. Chapter 1, by A. Hiltbrunner, F. Nagy and E. Schafer, starts by reviewing the long history leading to the identification of the phytochrome family and then mainly concentrates on photochemical properties and the molecular underpinnings of regulated subcellular phytochrome partitioning. In Chapter 2, A. Batschauer and colleagues review structure and function of the cryptochrome family and discuss recent insights into the molecular mechanism of the light-driven electron transfer within the molecules, nicely illustrated by a coloured scheme. The identification of interaction partners begins to shed some light on cryptochrome signalling. Chapter 3, by J. Christie, describes the phototropins and other LOV domain proteins. Particularly useful is the small comparative section on the well-characterized WHITE COLLAR and VIVID proteins in fungi and bacterial LOV-containing proteins. A short paragraph describing bacterial phytochromes would also have added to Chapter 1, since the connection between phytochromes and bacterial histidine kinases is mentioned en passant in later chapters. The second part of the book deals with selected aspects of photoreceptor signal transduction. Chapter 4, contributed by P. Quail, provides an excellent review of a suite of protein factors interacting with the phytochrome family members and the diverse biochemical and genetic means by which they have been identified. In particular, it resolves some of the conflicting interpretations on PHYTOCHROME INTERACTING FACTOR 3 function and how the field has moved from considering PIF3 as master regulator of phytochrome-mediated seedling de-etiolation to PIF3 fulfilling a more specialized function. Chapter 5, by C. Lillo et al., compiles information on the involvement of phosphorylation events in signalling downstream of the phytochrome, cryptochrome and phototropin photoreceptors. Appropriately the chapter ends with the notion that it will be important to integrate all these observations into a coherent network in the future. Chapter 6, contributed by S. Feng and X.W. Deng, introduces the reader to the ubiquitin/proteasome system and highlights its peculiarities as discovered in Arabidopsis, such as the enormous number of 1300 potential components of E3 ubiquitin ligases encoded in the genome. Subsequently it reviews the RING family E3 ubiquitin ligase COP1 that represses photomorphogenesis by promoting the degradation of key transcriptional activators. The chapter ends with an update on additional connections between light signalling and regulated proteolysis. In Chapter 7, G. Jenkins and B. Brown provide a snapshot on signalling events downstream of the UV-B light receptor, which still remain a mystery. They clearly illustrate the distinction between UV-B-triggered stress responses involving non-specific signalling and UV-B-triggered photomorphogenetic responses as a result of signalling events specific to the UV-B range. The chapter ends with a description of the genetic screens in search of the UV-B photoreceptor and of UVR8, the first signalling component of the UV-B-specific photomorphogenetic pathway. The third part of the book describes how selected physiological processes are governed by ambient light. P. Devlin reviews the pathways that promote the transition from vegetative to reproductive development, with special emphasis on the photoperiodic response resulting from the interaction of environmental light with the endogenous circadian clock. K. Franklin and G. Whitelam provide an excellent summary on the so-called shade-avoidance response that allows plants to use the ratio between red and far-red light to detect the presence of neighbours and accordingly adjust their development to escape shading canopies. Transcript profiling performed in the authors' laboratory has significantly advanced knowledge on signalling components mediating the shade-avoidance response, such as the basic helix–loop–helix transcription factors PIL1 and PIL2 (PIF3-like 1 and 2). E.-M. Josse and K. Halliday contribute a chapter on the interaction of photoreceptors with ‘other signals’. If space had permitted, each of these examples – including the circadian clock, phytohormones and ambient temperature – clearly would have deserved a chapter on its own. The last few years have witnessed significant advances in our understanding of the respective interwoven signalling networks, for example how auxin mediates bending of a shoot towards light, one of the classical observations in plant physiology. Particularly interesting is the last part of the book, devoted to applied aspects of photomorphogenesis that are not usually covered extensively in the standard updates on photoreceptors and light responses. M. Hudson discusses approaches to improve plant performance through manipulation of photoreceptors and reviews the use of the phytochrome system to establish a light-regulated gene expression system in yeast, developed by Quail and co-workers. Finally, N. Rajapakse and Y. Shahak describe light impact from a horticultural viewpoint. Overall, the authors provide a very useful update on all aspects of photoreceptor structure, signalling and their impact on plant physiology. Relevant examples from a plethora of species are covered, but data from Arabidopsis thaliana prevail by far. The beauty of the book lies in the fact that in most chapters new insights obtained by genome-wide molecular approaches (revealing that expression of about one-third of the Arabidopsis genes is different in light and dark) or physico-chemical techniques are integrated with a chronological perspective on photoreceptor function and signalling. Inevitably, one encounters some redundancy between individual contributions. Throughout, the literature cited provides a comprehensive resource of research papers and general reviews to consult for further details. The figures are well designed and informative and also well suited for teaching purposes. A few more photographs of, for example, mutant phenotypes would have been illustrative. Some of the chapters are in large parts suited for undergraduates with a background in plant molecular biology, and I highly recommend the book as a valuable read for graduate students and advanced researchers.

Flower formation in the long-day plant Sinapis alba is strictly dependent on an inductive light treatment. Differential screening of an apex cDNA library prepared 10 days after flower induction against cDNAs from vegetative apices has identified two cDNA clones, pSFD10.35 and pSFD10.44, which represent transcripts expressed transiently between day 10 and day 20 after induction and which disappear before flowers reach maturity. The corresponding full-length cDNAs which were isolated, Satap35 and Satap44, display 86% overall sequence identity. These cDNAs encode polypeptides with predicted molecular weights of 12.7 and 12.4 kDa, and isoelectric points of 10.4 and 7.5, respectively. The N-terminal portions of the open reading frames have characteristics of signal sequences. In situ hybridization reveals that both transcripts are localized exclusively in the tapetal cell layer of the anthers. Maximal expression is observed in flower buds of approximately 1.5 mm length (tetrad stage). Southern blot analysis demonstrates the presence of additional, closely related genes in the Sinapis genome, as well as the occurrence of homologous genes in Arabidopsis thaliana, Brassica napus and Nicotiana tabacum.

The activity of various light-regulated and developmentally regulated plant gene promoters critically depends upon the presence of a conserved sequence with a central CACGTG motif. Using band-shift assays, we have identified nuclear factor(s) from Nicotiana tabacum, termed CG-1, that specifically recognize(s) this transcriptional element in the ultraviolet-light-regulated Antirrhinum majus chalcone synthase promoter. CG-1 activity is constitutively expressed in tobacco seedlings grown in the absence of ultraviolet light as well as in seedlings induced for chalcone synthase gene expression by ultraviolet light irradiation. CG-1 activity has also been detected in flower tissue. DNA-protein cross-linking studies identified three polypeptides with apparent molecular masses of 20, 32 and 42 kDa binding to the CACGTG motif. Proteins interacting with the CACGTG motif were purified from N. tabacum seedlings using differential sequence-specific DNA affinity chromatography employing wild-type and mutated CG-1-binding sites. Denaturing polyacrylamide gel electrophoresis revealed major polypeptides of approximately 20, 30 and 40 kDa which are highly enriched in the affinity-purified fractions binding specifically to the CACGTG motif.

A positive regulatory element directing maximal expression of the Antirrhinum majus chalcone synthase promoter was characterized by protein-DNA-interaction studies and cis deletion analysis. The positive regulatory element consists of a 47 base pair direct repeat between positions -564 and -670 and provides three binding sites for nuclear protein factors from Nicotiana tabacum and Antirrhinum majus. Oligonucleotide competition assays revealed that the same factor(s) interact(s) with all three binding sites. Transient expression of chimeric chalcone synthase-neomycin phosphotransferase II genes in parsley protoplasts demonstrated that both halves of the 47 base pair repeat element are required for its in vivo function. A possible role of redundant binding sites for the positive regulatory function of the 47 base pair repeat element is discussed.