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Long non-coding RNAs (lncRNAs) have recently been found to widely exist in eukaryotes and play important roles in key biological processes. To extend our knowledge of lncRNAs in crop plants we performed both non-directional and strand-specific RNA-sequencing experiments to profile non-coding transcriptomes of various rice and maize organs at different developmental stages. Analysis of more than 3 billion reads identified 22 334 long intergenic non-coding RNAs (lincRNAs) and 6673 pairs of sense and natural antisense transcript (NAT). Many lincRNA genes were associated with epigenetic marks. Expression of rice lincRNA genes was significantly correlated with that of nearby protein-coding genes. A set of NAT genes also showed expression correlation with their sense genes. More than 200 rice lincRNA genes had homologous non-coding sequences in the maize genome. Much more lincRNA and NAT genes were derived from conserved genomic regions between the two cereals presenting positional conservation. Protein-coding genes flanking or having a sense-antisense relationship to these conserved lncRNA genes were mainly involved in development and stress responses, suggesting that the associated lncRNAs might have similar functions. Integrating previous genome-wide association studies (GWAS), we found that hundreds of lincRNAs contain trait-associated SNPs (single nucleotide polymorphisms [SNPs]) suggesting their putative contributions to developmental and agriculture traits., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Transposable elements (TEs) and repetitive sequences are ubiquitously present in eukaryotic genomes which are in general epigenetically silenced by DNA methylation and/or histone 3 lysine 9 methylation (H3K9me). RNA-directed DNA methylation (RdDM) is the major pathway that initiates de novo DNA methylation in Arabidopsis and sets up a self-reinforcing silencing loop between DNA methylation and H3K9me. However, a key issue is the requirement of a basal level transcript from the target loci to initiate the RNA-based silencing. How the heterochromatic silenced loci are transcribed remains largely unknown. Here, we show that JMJ24, a JmjC domain-containing protein counteracts H3K9me to promote basal level transcription of endogenous silenced loci in Arabidopsis. JMJ24 functionally resembles the fission yeast JmjC protein Epe1. The transcript promoted by JMJ24 is, at least in part, processed to small RNA to initiate the RdDM. Genome-wide transcriptome profiling indicates that transcript levels of TEs are more likely regulated by JMJ24, compared with protein-coding genes. Our data suggest that JMJ24 plays a conserved role in promoting basal level transcription of endogenous silenced loci to reinforce the silencing. We also provide evidence of a physical association between JMJ24 and RNA-dependent RNA polymerase 2 (RDR2), which represents an evolved property of the RNA silencing pathway., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

miR156 is an evolutionarily highly conserved miRNA in plants that defines an age-dependent flowering pathway. The investigations thus far have largely, if not exclusively, confined to plant aerial organs. Root branching architecture is a major determinant of water and nutrients uptake for plants. We show here that MIR156 genes are differentially expressed in specific cells/tissues of lateral roots. Plants overexpressing miR156 produce more lateral roots whereas reducing miR156 levels leads to fewer lateral roots. We demonstrate that at least one representative from the three groups of miR156 targets SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes: SPL3, SPL9 and SPL10 are involved in the repression of lateral root growth, with SPL10 playing a dominant role. In addition, both MIR156 and SPLs are responsive to auxin signaling suggesting that miR156/SPL modules might be involved in the proper timing of the lateral root developmental progression. Collectively, these results unravel a role for miR156/SPLs modules in lateral root development in Arabidopsis., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin-based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3-ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over-expression of isopentenyl transferase. Over-expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic-based selection systems, and may be used in combination with a negative selectable marker as part of a two-step selection system to obtain homoplasmic plant lines., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

The final expression level of a transgene-derived protein in transgenic plants depends on transcriptional and post-transcriptional processes. Here, we focus on methods to improve protein stability without comprising biological function. We found that the four isoforms of the Arabidopsis RAD23 protein family are relatively stable. The UBA2 domain derived from RAD23a can be used as a portable stabilizing signal to prolong the half-life of two unstable transcription factors (TFs), HFR1 and PIF3. The increased stability of the TF-UBA2 fusion proteins results in an enhanced phenotype in transgenic plants compared to expression of the TF alone. Similar results were obtained for the RAD23a UBA1 domain. In addition to UBA1/2 of RAD23a, the UBA domain from the Arabidopsis DDI1 protein also increased the half-life of the unstable protein JAZ10.1, which is involved in jasmonate signaling. Taken together, our results suggest that UBA fusions can be used to increase the stability of unstable proteins for basic plant biology research as well as crop improvement., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

RNA silencing is an important antiviral mechanism in diverse eukaryotic organisms. In Arabidopsis DICER-LIKE 4 (DCL4) is the primary antiviral Dicer, required for the production of viral small RNAs from positive-strand RNA viruses. Here, we showed that DCL4 and its interacting partner dsRNA-binding protein 4 (DRB4) participate in the antiviral response to Turnip yellow mosaic virus (TYMV), and that both proteins are required for TYMV-derived small RNA production. In addition, our results indicate that DRB4 has a negative effect on viral coat protein accumulation. Upon infection DRB4 expression was induced and DRB4 protein was recruited from the nucleus to the cytoplasm, where replication and translation of viral RNA occur. DRB4 was associated with viral RNA in vivo and directly interacted in vitro with a TYMV RNA translational enhancer, raising the possibility that DRB4 might repress viral RNA translation. In plants the role of RNA silencing in viral RNA degradation is well established, but its potential function in the regulation of viral protein levels has not yet been explored. We observed that severe infection symptoms are not necessarily correlated with enhanced viral RNA levels, but might be caused by elevated accumulation of viral proteins. Our findings suggest that the control of viral protein as well as RNA levels might be important for mounting an efficient antiviral response., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

In plants, the exogenous transgene transcribing inverted-repeat (exo-IR) sequences produces double-stranded RNAs that are processed by DCL4. The 21-nt small interfering RNAs generated function as mobile signals to trigger non-cell autonomous silencing of target endogenes in the neighboring 10-15 cells. The potential involvement of nuclear silencing pathway components in signal spreading or sensing in target cells is not clear. Here, we demonstrate that the exo-IR silencer (exo-Pdsi) is negatively autoregulated through methylation spreading, which acts in cis to reinforce the self-silencing of the silencer. Mutations affecting nuclear proteins DRD1 and Pol V (NRPE1 or NRPD2) relieved exo-Pdsi self-silencing, resulting in higher levels of Pdsi transcripts, which increased the non-cell autonomous silencing of endo-PDS. Our results suggest that in an experimental silencing pathway, methylation spreading on a silencer transgene may not have a direct endogenous plant counterpart when the protein-encoding gene is the target. DRD1-Pol V-dependent de novo methylation, by acting in cis to reinforce self-silencing of exo-IR, may play a role in restraining the inappropriate silencing of active protein-coding genes in plants., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24 nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Ubiquitination plays important roles in plant development, including programmed cell death. Here, we characterize a novel membrane-bound RING motif protein, encoded by RING1, that is expressed at a low level in all Arabidopsis tissues but can be upregulated by fumonisin B1 (FB1) treatment and pathogen infection. RING1 displays E3 ubiquitin ligase activity in vitro, which is dependent on the integrity of the RING motif. GFP fusion protein localization and cell fractionation experiments show that this E3 ligase is associated with the lipid rafts of plasma membranes. Knock-down of RING1 transcripts using artificial microRNA (amiR-R1(159)) leads to FB1 hyposensitivity, but overexpression of RING1 confers hypersensitivity. Additionally, expression of the pathogenesis-related 1 (PR-1) gene is lower and delayed in amiR-R1(159) plants compared with wild-type and RING1-overexpressing plants. The FB1 hyposensitivity of amiR-R1(159) plants can be rescued by expression of cleavage-resistant RING1mut transcripts. Our results suggest that RING1 acts as a signal from the plasma membrane lipid rafts to trigger the FB1-induced plant programmed cell death pathway.

Upon seed imbibition, abscisic acid (ABA) levels decrease to allow embryos to germinate and develop into seedlings. However, under abiotic stress conditions, ABA levels remain high, and growth and development are arrested. Several transcription factors, including abscisic acid-insensitive (ABI)3 and ABI5, are known to control this developmental checkpoint. Here, we show that, in germinating Arabidopsis thaliana seeds, ABA induces the accumulation of microRNA 159 (miR159) in an ABI3-dependent fashion, and miRNA159 mediates cleavage of MYB101 and MYB33 transcripts in vitro and in vivo. The two MYB transcription factors function as positive regulators of ABA responses, as null mutants of myb33 and myb101 show hyposensitivity to the hormone. Consistent with this, miR159 over-expression suppresses MYB33 and MYB101 transcript levels and renders plants hyposensitive to ABA, whereas transgenic plants over-expressing cleavage-resistant forms of MYB33 and MYB101 are hypersensitive, as are plants over-expressing the Turnip mosaic virus (TuMV) P1/HC-Pro viral protein that is known to inhibit miRNA function. Our results suggest that ABA-induced accumulation of miR159 is a homeostatic mechanism to direct MYB33 and MYB101 transcript degradation to desensitize hormone signaling during seedling stress responses.

Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1-119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth.

Arabidopsis phytochrome A (phyA) regulates not only seed germination and seedling de-etiolation but also circadian rhythms and flowering time in adult plants. The SUPPRESSOR OF PHYA-105 (SPA1) acts as a negative regulator of phyA-mediated de-etiolation of young seedlings, but its roles in adult plants have not yet been described. Here, we show that SPA1 is involved in regulating circadian rhythms and flowering time in plants. Under constant light, the abundance of SPA1 protein exhibited circadian regulation, whereas under constant darkness, SPA1 protein levels remained unchanged. These results indicate that the SPA1 protein is controlled by the circadian clock and light signals. In addition, the spa1-3 mutation slightly shortened the circadian period of CCA1, TOC1/PRR1 and SPA1 transcript accumulation under constant light. Phenotypic analysis showed that the spa1-3 mutant flowers early under short-day (SD) but not long-day (LD) conditions. Consistent with this finding, transcripts encoding flowering locus T (FT), which promotes flowering, increased in spa1-3 under only SD conditions, although the CONSTANS (CO) transcript level was not affected under either SD nor LD conditions. Our results indicate that SPA1 not only negatively controls phyA-mediated signaling in seedlings, but also regulates circadian rhythms and flowering time in plants.

Phytochrome A (phyA) plays a primary role in initiating seedling de-etiolation and is the only plant photoreceptor known to be activated by far-red light (FR). The signaling intermediate FHY1 appears to either participate directly in relaying the phyA signal or to positively regulate a critical signaling event(s) downstream of phyA activation. Here we identify a homolog of FHY1 named FHL (FHY1-like) as a novel signaling factor essential for complete responsiveness to phyA. FHL possesses functional nuclear localization and nuclear export signals. Lines in which FHL function was abolished by insertional mutagenesis or attenuated by RNAi-mediated suppression displayed a weaker hyposensitivity to continuous FR than fhy1 null mutants and most reported phyA signaling mutants. However, hypocotyl elongation assays indicated that suppression of FHL expression in fhy1-3 caused an insensitivity of hypocotyl elongation to FR and blue light (B) indistinguishable from that seen in phyA. Real-time PCR indicates that in FR, FHY1 transcripts are approximately 15-fold more abundant than FHL transcripts. Although both FHY1 and FHL are capable of homo- and hetero-interaction via their C-termini, the ability of FHL overexpression to restore wild-type (WT) morphological and molecular phenotypes to fhy1-3 seedlings suggests that the extreme insensitivity to FR associated with suppression of FHL expression in fhy1-3 cannot be accounted for by a critical role for FHY1-FHL heterodimers in phyA signal transmission. Rather, we suggest that the relative abundances of FHY1 and FHL in WT plants account for the differences in the severity of fhy1 and fhl mutations. As for FHY1, FHL transcript accumulation is dependent on FHY3 and is decreased after exposure to FR, R or B light. These findings reiterate the prevalence of partial degeneracy in plant signaling networks that regulate responses crucial to survival.

Class III homeodomain-leucine zipper proteins regulate critical aspects of plant development, including lateral organ polarity, apical and lateral meristem formation, and vascular development. ATHB15, a member of this transcription factor family, is exclusively expressed in vascular tissues. Recently, a microRNA (miRNA) binding sequence has been identified in ATHB15 mRNA, suggesting that a molecular mechanism governed by miRNA binding may direct vascular development through ATHB15. Here, we show that miR166-mediated ATHB15 mRNA cleavage is a principal mechanism for the regulation of vascular development. In a gain-of-function MIR166a mutant, the decreased transcript level of ATHB15 was accompanied by an altered vascular system with expanded xylem tissue and interfascicular region, indicative of accelerated vascular cell differentiation from cambial/procambial cells. A similar phenotype was observed in Arabidopsis plants with reduced ATHB15 expression but reversed in transgenic plants overexpressing an miR166-resistant ATHB15. ATHB15 mRNA cleavage occurred in standard wheat germ extracts and in Arabidopsis and was mediated by miR166 in Nicotiana benthamiana cells. miR166-assisted ATHB15 repression is likely to be a conserved mechanism that regulates vascular development in all vascular plants.

Plants use the family of phytochrome photoreceptors to sense their light environment in the red/far-red region of the spectrum. Phytochrome A (phyA) is the primary photoreceptor that regulates germination and early seedling development. This phytochrome mediates seedling de-etiolation for the developmental transition from heterotrophic to photoauxotrophic growth. High intensity far-red light provides a way to specifically assess the role of phyA in this process and was used to isolate phyA-signaling intermediates. fhy1 and pat3 (renamed fhy1-3) are independently isolated alleles of a gene encoding a phyA signal transduction component. FHY1 is a small 24 kDa protein that shows no homology to known functional motifs, besides a small conserved septin-related domain at the C-terminus, a putative nuclear localization signal (NLS) and a putative nuclear exclusion signal (NES). Here we demonstrate that the septin-related domain is important for FHY1 to transmit phyA signals. Moreover, the putative NLS and NES of FHY1 are indeed involved in its nuclear localization and exclusion. Nuclear localization of FHY1 is needed for it to execute responses downstream of phyA. Together with the results from global expression analysis, our findings point to an important role of FHY1 in phyA signaling through its nuclear translocation and induction of gene expression.

Cyclic ADP-ribose (cADPR) was previously shown to activate transient expression of two abscisic acid (ABA)-responsive genes in tomato cells. Here, we show that the activity of the enzyme responsible for cADPR synthesis, ADP-ribosyl (ADPR) cyclase, is rapidly induced by ABA in both wild-type (WT) and abi1-1 mutant Arabidopsis plants in the absence of protein synthesis. Furthermore, in transgenic Arabidopsis plants, induced expression of the Aplysia ADPR cyclase gene resulted in an increase in ADPR cyclase activity and cADPR levels, as well as elevated expression of ABA-responsive genes KIN2, RD22, RD29a, and COR47 (although to a lesser extent than after ABA induction). Genome-wide profiling indicated that about 28% of all ABA-responsive genes in Arabidopsis are similarly up- and downregulated by cADPR and contributed to the identification of new ABA-responsive genes. Our results suggest that activation of ADPR cyclase is an early ABA-signaling event partially insensitive to the abi1-1 mutation and that an increase in cADPR plays an important role in downstream molecular and physiological ABA responses.

We report the characterization of a plant gene encoding a member of the BET subgroup of bromodomain proteins, a novel class of putative transcription factors. Imbibition-inducible 1 (IMB1) appears to be a nuclear protein as suggested by subcellular localization in onion epidermal cells using an IMB1-yellow fluorescent protein (YFP) fusion protein. In Arabidopsis thaliana, IMB1 is expressed at very low levels in dry seeds, but is markedly induced during seed imbibition. In addition, IMB1 transcript levels are down regulated during germination. Seeds of a loss-of-function mutant allele, imb1, show impaired cotyledon greening during germination in abscisic acid (ABA) and express higher levels of ABI5 protein than the wild type. Moreover, imb1 seeds are deficient in the phytochrome A (phyA)-mediated very-low-fluence response of germination. Microarray analysis revealed that genes included in different functional categories, such as cell-wall metabolism or plastid function, are repressed in imbibed imb1 seeds. Mutant imb1 plants appear normal, indicating that IMB1 is involved in regulating a specific developmental stage. Taken together, these results show that IMB1 plays a role in the promotion of seed germination by both negatively and positively regulating the ABA and phyA transduction pathways, respectively. In imbibed seeds, IMB1 modulates the transcription of a battery of genes, providing clues on its mode of action.

Constitutive expression of an intron-containing self-complementary 'hairpin' RNA (ihpRNA) has recently been shown to efficiently silence target genes in transgenic plants. However, this technique cannot be applied to genes whose silencing may block plant regeneration or result in embryo lethality. To obviate these potential problems, we have used a chemical-inducible Cre/loxP (CLX) recombination system to trigger the expression of an intron-containing inverted-repeat RNA (RNAi) in plants. A detailed characterization of the inducible RNAi system in transgenic Arabidopsis thaliana and Nicotiana benthamiana plants demonstrated that this system is stringently controlled. Moreover, it can be used to induce silencing of both transgenes and endogenous genes at different developmental stages and at high efficiency and without any detectable secondary affects. In addition to inducing complete silencing, the RNAi can be produced at various times after germination to initiate and obtain different degrees of gene silencing. Upon induction, transgenic plants with genetic chimera were obtained as demonstrated by PCR analysis. Such chimeric plants may provide a useful system to study signaling mechanisms of gene silencing in Arabidopsis as well as other cases of long-distance signaling without grafting. The merits of using the inducible CLX system for RNAi expression are discussed.

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.

We have developed a new chemical inducible genetic system that allows for the isolation of any cDNA molecule from in vitro generated genomic transgenes in transgenic plants. This system, termed regulated in vivo cDNA generation (RIDE), permits both targeted isolation of individual full-length cDNA molecules and random isolation of any partial or full-length cDNA from in planta genomic libraries. The RIDE system makes use of the 17-beta estradiol-inducible promoter system linked to intron donor and acceptor sites in a new binary vector configuration. In transgenic Arabidopsis and tobacco plants, we show that the RIDE system can isolate low-abundance full-length cDNAs previously unattainable by conventional means at high efficiencies (75-85%). The ability to randomly isolate individual exons and exons spliced together from genomic libraries in planta suggest that this system can be used for the isolation of any cDNA molecules. The RIDE system thus appears to be an efficient and versatile system for the generation of potentially any cDNA molecule. Moreover, the ORF structural data generated will be of value in both verifying and correcting computational ORF predications in the databases available to the scientific community.

The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

In plant cells, plastids divide by binary fission involving a complex pathway of events. Although there are clear similarities between bacterial and plastid division, limited information exists regarding the mechanism of plastid division in higher plants. Here we demonstrate that AtMinE1, an Arabidopsis homologue of the bacterial MinE topological specificity factor, is an essential integral component of the plastid division machinery. In prokaryotes MinE imparts topological specificity during cell division by blocking division apparatus assembly at sites other than midcell. We demonstrate that overexpression of AtMinE1 in E. coli results in loss of topological specificity and minicell formation suggesting evolutionary conservation of MinE mode of action. We further show that AtMinE1 can indeed act as a topological specificity factor during plastid division revealing that AtMinE1 overexpression in Arabidopsis seedlings results in division site misplacement giving rise to multiple constrictions along the length of plastids. In agreement with cell division studies in bacteria, AtMinE1 and AtMinD1 show distinct intraplastidic localisation patterns suggestive of dynamic localisation behaviour. Taken together our findings demonstrate that AtMinE1 is an evolutionary conserved topological specificity factor, most probably acting in concert with AtMinD1, required for correct plastid division in Arabidopsis.

The interaction of phytochrome signalling with the SA signal transduction pathway has been investigated in Arabidopsis using single and multiple mutants affected in light perception (phyA and phyB deficient) and light-signal processing (psi2, phytochrome signalling). The induction of PR1 by SA and functional analogues has been found to strictly correlate with the activity of the signalling pathway controlled by both phyA and phyB photoreceptors. In darkness as well as dim light, and independently of a carbohydrate source, SA-induced PR gene expression as well as the hypersensitive response to pathogens (HR) are strongly reduced. Moreover, the initiation of HR also exhibits a strict dependence upon both the presence and the amplitude of a phytochrome-elicited signal. The growth of an incompatible strain of bacterial a pathogen (Pseudomonas syringae pv. tomato) was enhanced in phyA-phyB and decreased in psi2 mutants. While functional chloroplasts were found necessary for the development of an HR, the induction of PRs was strictly dependent on light, but independent of functional chloroplasts. Taken together, these data demonstrate that the light-induced signalling pathway interacts with the pathogen/SA-mediated signal transduction route. These results are summarized in a formalism that allows qualitative computer simulation.

Formation of somatic embryos in plants is known to require high concentrations of auxin or 2,4-dichlorophenoxyacetic acid (2,4-D), which presumably acts to trigger a signalling cascade. However, very little is known about the molecular mechanism that mediates the vegetative-to-embryogenic transition. We have employed a genetic approach to dissect the signal transduction pathway during somatic embryogenesis. In a functional screen using a chemical-inducible activation-tagging system, we identified two alleles of Arabidopsis gene PGA6 whose induced overexpression caused high-frequency somatic embryo formation in all tissues and organs tested, without any external plant hormones. Upon inducer withdrawal, all these somatic embryos were able to germinate directly, without any further treatment, and to develop into fertile adult plants. PGA6 was found to be identical to WUSCHEL (WUS), a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and floral meristems. Transgenic plants carrying an estradiol-inducible XVE-WUS transgene can phenocopy pga6-1 and pga6-2. Our results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.

Different alpha-tubulin cDNA sequences fused in an antisense orientation to a CaMV 35S promoter were introduced into Arabidopsis thaliana plants. Several independent transgenic lines that showed a moderate but clear reduction of alpha-tubulin gene expression (TUA6/AS lines) were obtained and phenotypically characterized. Although no apparent abnormalities were detected in the aerial parts of TUA6/AS plants, root development was severely affected. Cells in TUA6/AS root tips were found to contain aberrant microtubular structures, to expand abnormally and to be unable to undergo regular cell division. These cellular defects caused a dramatic radial expansion of the root tip and inhibited root elongation. In addition, TUA6/AS roots displayed ectopic formation of root hairs, root hair branching and a reduced ability to respond to gravitropic challenges. Our results contribute to an improved understanding of the different roles microtubules play during root development and demonstrate that reverse genetics is a powerful tool to analyze cytoskeletal functions during plant organogenesis.

We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the -46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.

The physiological acclimation of plants to osmotic stresses involves a complex programme of gene regulation. In one signalling pathway, elevated levels of abscisic acid (ABA) activate a subset of stress genes. Because ABA responses lack a definable morphological phenotype, we have screened for mutants that exhibit deregulated ABA-responsive gene expression. To monitor this ABA response, a line of Arabidopsis thaliana carrying a transgene composed of the ABA-responsive Arabidopsis kin2 promoter fused to the coding sequence for the firefly luciferase gene, kin2::luc, was generated. Patterns of ABA-responsive luciferase activity were monitored by photon counting. In contrast to wild-type plants which display a transient activation of kin2::luc, an ABA deregulated gene expression mutant (ade1) exhibits both sustained and enhanced levels of transgene activity. Levels of kin2, cor47 and rab18 expression in ade1 plants are also enhanced and prolonged indicating that the molecular mechanism(s) altered in ade1 plants affects the regulation of other ABA-responsive genes. The mutant phenotype is specific for the ABA response as cold-inducible kin2 expression is unaltered in ade1 plants. Genetic analyses indicate that the ade1 mutant is a monogenic recessive trait. A role for negative regulator function in ABA signalling is discussed.

The C-terminus of mouse talin (amino acids 2345-2541) is responsible for all of the protein's f-actin binding capacity. Unlike full-length talin, the C-terminal f-actin binding domain is unable to nucleate actin polymerization. We have found that transient and stable expression of the talin actin-binding domain fused to the C-terminus of the green fluorescent protein (GFP-mTn) can visualize the actin cytoskeleton in different types of living plant cells without affecting cell morphology or function. Transiently expressed GFP-mTn co-localized with rhodamine-phalloidin in permeabilized tobacco BY-2 suspension cells, showing that the fusion protein can specifically label the plant actin cytoskeleton. Constitutive expression of GFP-mTn in transgenic Arabidopsis thaliana plants visualized actin filaments in all examined tissues with no apparent effects on plant morphology or development at any stage during the life cycle. This demonstrates that in a number of different cell types GFP-mTn can serve as a non-invasive marker for the actin cytoskeleton. Confocal imaging of GFP-mTn labeled actin filaments was employed to reveal novel information on the in vivo organization of the actin cytoskeleton in transiently transformed, normally elongating tobacco pollen tubes.

Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid-inducible promoter. Dexamethasone-induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae-induced hypersensitive response and also induced the expression of a pathogenesis-related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2-101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid-inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene-specified cell death response in both resistant and susceptible plants.

Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown. A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals. For this purpose, an in vivo reporter system was established consisting of the light- and sugar-regulated plastocyanin promoter, fused to the luciferase coding sequence (PC-LUC construct). At the seedling stage, expression of the PC-LUC gene is repressed by sucrose, and a number of sucrose-uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter. Three mutants have been characterized in more detail. The sugar analog 2-deoxy-D-glucose (2DG) was used to repress whole plant photosynthesis, PC-LUC gene expression and total ribulose-1,5-bisphosphate activity. It was found that the sun6 mutation makes plants unresponsive to these 2DG-induced effects. Moreover, unlike wild-type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium. These findings suggest that the SUN6 gene is active in a hexose-activated signal transduction pathway.

A novel chemical induction system for transcription in plants has been developed, taking advantage of the regulatory mechanism of vertebrate steroid hormone receptors. A chimeric transcription of the DNA-binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR). The GVG gene was introduced into transgenic tobacco and Arabidopsis together with a luciferase (Luc) gene which was transcribed from a promoter containing six tandem copies of the GAL4 upstream activating sequence. Induction of luciferase activity was observed when the transgenic tobacco plants were grown on an agar medium containing dexamethasone (DEX), a strong synthetic glucocorticoid. Induction levels of the luciferase activity were well correlated with DEX concentrations in the range from 0.1 to 10 microM and the maximum expression level was over 100 times that of the basal level. Analysis of the induction kinetics by Northern blot analysis showed that the Luc mRNA was first detected 1 h after DEX treatment and increased to the maximum level in 4 h. The stationary induction level and the duration of the induction varied with the glucocorticoid derivative used. The GVG gene activity can also be regulated by DEX in transgenic Arabidopsis plants. The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system. Advantages and potential uses of this system are also discussed.

Previous work using microinjection into single cells of the tomato aurea mutant demonstrated that phytochrome A-dependent activation of rbcS and chs genes was mediated by calcium and cGMP, respectively. This work sought to identify promoter cis-elements that respond to these two small molecules. Box II and Unit I, derived from rbcS-3A and chs promoters, respectively, were previously shown to function as light-responsive cis-elements. Eleven copies of Box II and four copies of Unit I were linked 5' to the -90 and -46 35 S promoters, respectively, and, both constructs were fused to the beta-glucuronidase (GUS) reporter gene. GUS activities were obtained upon coinjection of either Box II/-90GUS or Unit I/-46GUS with oat phytochrome A (phyA) and GTP gamma S, an activator of heterotrimeric G proteins. The activation of Box II/-90GUS by phyA was insensitive to the cGMP antagonist, Rp-cGMPS, although anthocyanin accumulation, but not chloroplast development, was totally blocked in the injected cells. Consistent with this result, calcium, but not cGMP, induced Box II/-90GUS activity. In contrast to Box II/-90GUS, phyA-dependent activation of Unit I/-46GUS activity was blocked by Rp-cGMPS. Moreover, cGMP, not calcium, induced Unit I/-46GUS activity. Control experiments showed that -90 GUS and -46 GUS were inactive in the presence of calcium and cGMP, respectively. These results provide evidence that Box II and Unit I are targets of the calcium and cGMP pathways, respectively. Interestingly, calcium activation of Box II/-90GUS was repressed by a high concentration of cGMP and cGMP induction of Unit I/-46GUS was blocked by a high concentration of calcium/CaM. Thus, these two small cis-elements can also serve as targets of the reciprocal control mechanisms that operate to regulate the activities of the two phyA signaling branches.

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome-regulated genes in fhy1. In far-red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A-deficient mutant, confirming that gene expression in far-red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light-induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A-mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue-light signaling pathway for CHS expression.

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase (chs) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA, phyB) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as beta-glucuronidase (GUS) expression mediated by light-regulated promoters (chs, chlorophyll a/b binding protein (lhcb1) and ferredoxin NADP+ oxidoreductase (fnn). Microinjection of phyA under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs-GUS, lhcb 1-GUS and fnr-GUS expression. Microinjection of phyB under identical conditions induced chlorophyll accumulation and mediated lhcb 1-GUS and fnr-GUS expression but neither anthocyanin accumulation nor chs-GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB injections under red light conditions indicated that phyB needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal.

The fission yeast Schizosaccharomyces pombe has been used to identify Arabidopsis thaliana proteins that may play a role in cell shape maintenance or cell cycle regulation. An Arabidopsis thaliana cDNA library was constructed in pREP5N vector under the control of the inducible nmt1 promoter and transformed into S. pombe. Expression of the A. thaliana sequences was induced and clones showing severe morphological changes were identified and analysed. Comparison of the sequences of the inserts with the sequence data bases revealed that several cDNAs encode proteins known to play a role in function of the cytoskeleton, the cell cycle and establishment of cell polarity. These include alpha-1, alpha-2, alpha-6 and beta-6 tubulins, myosin heavy chain-like protein, ubiquitin conjugating enzymes UBC9 (E2), 26S protease subunits, Ranbinding protein, myb protein, PRL1 gene product and rho protein. Approximately 30% of the clones encode novel sequences. The results suggest that S. pombe phenotypic screening can be used to identify plant proteins involved in cell shape maintenance and regulation during cell cycle and development.

Four members of the Arabidopsis profilin (pfn) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group (pfn1 and pfn2) is expressed in all organs of the plant and the other group (pfn3 and pfn4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71-75% identical. The Arabidopsis profilins bind poly-L-proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn4 is restricted to mature and germinating pollen grains.

Previously, it has been shown that a trimer of a 22 bp fragment of the promoter of the seed-specific pea lectin gene confers high expression in seed. Here it is reported that this fragment contains a binding site for the cloned basic domain/leucine zipper (bZIP) proteins TGA1a and Opaque-2 (O2). Gel shift assays, DNasel footprinting and methylation interference assays using purified TGA1a were performed to determine whether additional binding sites are present in the psl promoter. Within the 469 bp upstream region only one other TGA1a binding site was found, which is much weaker than the one present in the 22 bp element. Both O2 and TGA1a bound to the odd base palindromic C-box sequence, ATGAGTCAT, present within the 22 bp fragment. The 22 bp fragment also contains the sequence CACGTA, which contains the ACGT core usually found in binding sites for bZIP proteins. However, this sequence did not significantly contribute to bZIP protein binding. The binding affinity of TGA1a for the odd base palindromic sequence was low relative to a high-affinity C-box (ATGACGTCAT). By contrast, O2 strongly bound to the odd base C-box; the affinity was comparable with that for high-affinity G-(GACACGTGTC) and C-boxes. It is concluded that the presence of an ACGT core sequence is not a prerequisite for high-affinity binding of O2.

A single copy gene has been isolated, termed GOS2, from rice. Sequence comparison revealed highly similar genes in mammals and yeast, indicating that GOS2 encodes an evolutionary conserved protein. GOS2 mRNA was detected in all tissues examined. When the upstream region was translationally fused to the reporter gene gusA it was found to drive expression in a variety of rice tissues and in cell suspensions of other monocot species following introduction by particle bombardment. Therefore, the GOS2 promoter is potentially useful for genetic engineering of monocots. A DNA-binding activity from rice, termed rice ASF-1, with similar binding specificity as the cloned tobacco transcription factor TGA-1a, was found to bind to a TGACG sequence motif in the GOS2 promoter. Possible roles for rice ASF-1 in the transcriptional activation of the GOS2 promoter are discussed.