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Light signals perceived by phytochromes (Phys) and cryptochromes (Crys) play key roles in plant growth and development and in photoperiod dependant process such as flowering, tuberization, seasonal growth cessation and dormancy. The integration of the light signals with the endogenous circadian oscillator provides plants with a mechanism to monitor changes in photo-period or day-length. In a recent report, we established that in Vitis vinifera L. cv Thompson Seedless, photoperiod drives the entrance of buds into endodormancy (ED) and modifies the expression of VvPHYA and VvPHYB transcripts in grapevine leaves, suggesting that both VvPHYs could play crucial roles in SD-induced transition of bud into ED. Here, we aimed to establish whether the transition of grapevine buds into ED is a mere consequence of a decision taken in the leaf or whether the bud responds by itself to photoperiod. Results show that in defoliated grapevine canes, bud-ED development is delayed compared with non-defoliated control canes, and that under LD-photoperiod both VvPHYA and VvPHYB transcripts are highly expressed in grapevine buds, whilst under SD-photoperiod both VvPHYs are downregulated and expression can not be detected. Overall, the results suggest that grapevine bud behaves as semi-autonomous organ in sensing the photoperiod signal, and that VvPHYA and VvPHYB gene expression is differently regulated by photoperiod in leaf and bud of grapevines.

The Arabidopsis flowering locus T (FT) gene encodes the mobile florigen essential for floral induction. While movement of the FT protein has been shown to occur within plants, systemic spread of FT mRNA remains to be unequivocally demonstrated. Utilizing novel RNA mobility assay vectors based on two distinct movement-defective viruses, Potato virus X and Turnip crinkle virus, and an agroinfiltration assay, we demonstrate that nontranslatable FT mRNA, independent of the FT protein, moves throughout Nicotiana benthamiana and mutant Arabidopsis plants and promotes systemic trafficking of viral and green fluorescence protein RNAs. Viral ectopic expression of FT induced flowering in the short-day N. tabacum Maryland Mammoth tobacco under long-day conditions. Recombinant Potato virus X bearing FT RNA spread and established systemic infection more quickly than the parental virus. The cis-acting element essential for RNA movement was mapped to the nucleotides 1 to 102 of the FT mRNA coding sequence. These data demonstrate that a plant self-mobile RNA molecule can mediate long-distance trafficking of heterologous RNAs and raise the possibility that FT RNA, along with the FT protein, may be involved in the spread of the floral stimulus throughout the plant.

Phytochromes are red (R)/far-red (FR) light photoreceptors that play fundamental roles in photoperception of the light environment and the subsequent adaptation of plant growth and development. There are five distinct phytochromes in Arabidopsis thaliana, designated phytochrome A (phyA) to phyE. phyA is light-labile and is the primary photoreceptor responsible for mediating photomorphogenic responses in FR light, whereas phyB-phyE are light stable, and phyB is the predominant phytochrome regulating de-etiolation responses in R light. Phytochromes are synthesized in the cytosol in their inactive Pr form. Upon light irradiation, phytochromes are converted to the biologically active Pfr form, and translocate into the nucleus. phyB can enter the nucleus by itself in response to R light, whereas phyA nuclear import depends on two small plant-specific proteins FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL). Phytochromes may function as light-regulated serine/threonine kinases, and can phosphorylate several substrates, including themselves in vitro. Phytochromes are phosphoproteins, and can be dephosphorylated by a few protein phosphatases. Photoactivated phytochromes rapidly change the expression of light-responsive genes by repressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), an E3 ubiquitin ligase targeting several photomorphogenesis-promoting transcription factors for degradation, and by inducing rapid phosphorylation and degradation of Phytochrome-Interacting Factors (PIFs), a group of bHLH transcription factors repressing photomorphogenesis. Phytochromes are targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway.

Flowering is one of the most important steps in a plant life cycle. Plants utilize light as an informational source to determine the timing of flowering. In Arabidopsis, phytochrome A (phyA), phyB and cryptochrome2 (cry2) are major photoreceptors that regulate flowering. These photoreceptors perceive light stimuli by leaves for the regulation of flowering. A leaf is an organ consisting of different tissues such as epidermis, mesophyll and vascular bundles. In the present study, we examined in which tissue the light signals are perceived and how those signals are integrated within a leaf to regulate flowering. For this purpose, we established transgenic Arabidopsis lines that expressed a phyB-green fluorescent protein (GFP) fusion protein or a cry2-GFP fusion protein in organ/tissue-specific manners. Consequently, phyB was shown to perceive light stimuli in mesophyll. By contrast, cry2 functioned only in vascular bundles. We further confirmed that both phyB-GFP and cry2-GFP regulated flowering by altering the expression of a key flowering gene, FT, in vascular bundles. In summary, perception sites for different spectra of light are spatially separated within a leaf and the signals are integrated through the inter-tissue communication.

The phytochrome (phy) photoreceptor family regulates almost all aspects of plant development in a broad range of light environments including seed germination, onset of the photomorphogenic program in seedling stage, the shade avoidance syndrome in competing plant communities, flowering induction and senescence of adult plants. During evolution two clearly distinct classes of phy-s emerged covering these very different physiological tasks.1 PhyA is rapidly degraded in its activated state. PhyA functions in controlling seed germination at very low light intensities (very low fluence response, VLFR) and seedling establishment under photosynthetic shade conditions (high irradiance response, HIR) where the far-red portion of the transmitted light to understorey habitats is substantially enhanced. Arabidopsis phyB together with phyC, D and E belongs to the relatively stable sensor class in comparison to the light labile phyA. PhyB functions at all stages of development including seed germination and seedling establishment, mediates classical red/far-red reversible low fluence responses (LFR) as well as red light high irradiance responses, and it is considered to be the dominating phytochrome sensor of its class.

Phytochromes sense red/far-red light and trigger a cascade of physiological responses in plant. Here, a phytochrome B homolog, GmPHYB1, was amplified from the soybean genome, and its expression profiles were obtained for various parts of the plant and at various developmental stages. The gene was ectopically expressed in Arabidopsis thaliana, driven by CaMV 35S promoter, to study the physiological functions of the gene product. The overexpressors of GmPHYB1 behaved similarly to those of AtPHYB, but with some subtle differences with respect to the acceleration of flowering under short day conditions and the growth of the hypocotyl under certain light fluence rate. The results suggested that this soybean PHYB homolog was well conserved both at the level of sequence and physiological function.

Summary

The synergism between red and blue light in the control of plant growth and development [1, 2] requires the co-action of the red/far-red light photoreceptor phytochrome B (phyB) and the blue/UV-A receptors, cryptochromes (cry) [3]. Here we describe the mechanism for the co-action of these photoreceptors in controlling both development and physiology. In seedlings grown under red light, a transient supplement with blue light induced persistent changes in the transcriptome and growth patterns. Blue light enhanced the expression of the transcription factors LONG HYPOCOTYL 5 (HY5) and HOMOLOG OF HY5 (HYH) [4] and of SUPPRESSOR OF PHYA 1 (SPA1) and SPA4 [5]. HY5 and HYH enhanced phyB signalling output beyond the duration of the blue-light signal and, contrary to their known role as repressors of phyA signalling [5], SPA1 and SPA4 also enhanced phyB signalling. These observations demonstrate that the mechanism of synergism involves the promotion by cry of positive regulators of phyB signalling. The persistence of the light-derived signal into the night commits the seedling to a morphogenetic and physiological program consistent with a photosynthetic lifestyle.

In inducing photoperiodic conditions, plants produce a signal dubbed “florigen” in leaves. Florigen moves through the phloem to the shoot apical meristem (SAM) where it induces flowering. In Arabidopsis, the FLOWERING LOCUS T (FT) protein acts as a component of this phloem-mobile signal. However whether the transportable FT mRNA also contributes to systemic florigen signalling remains to be elucidated. Using non-conventional approaches that exploit virus-induced RNA silencing and meristem exclusion of virus infection, we demonstrated that the Arabidopsis FT mRNA, independent of the FT protein, can move into the SAM. Viral ectopic expression of a non-translatable FT mRNA promoted earlier flowering in the short-day (SD) Nicotiana tabacum Maryland Mammoth tobacco in SD. These data suggest a possible role for FT mRNA in systemic floral signalling, and also demonstrate that cis-transportation of cellular mRNA into SAM and meristem exclusion of pathogenic RNAs are two mechanistically distinct processes.

Background

Plants have evolved various sophisticated mechanisms to respond and adapt to changes of abiotic factors in their natural environment. Light is one of the most important abiotic environmental factors and it regulates plant growth and development throughout their entire life cycle. To monitor the intensity and spectral composition of the ambient light environment, plants have evolved multiple photoreceptors, including the red/far-red light-sensing phytochromes.

Methodology/Principal Findings

We have developed an integrative mathematical model that describes how phytochrome B (phyB), an essential receptor in Arabidopsis thaliana, controls growth. Our model is based on a multiscale approach and connects the mesoscopic intracellular phyB protein dynamics to the macroscopic growth phenotype. To establish reliable and relevant parameters for the model phyB regulated growth we measured: accumulation and degradation, dark reversion kinetics and the dynamic behavior of different nuclear phyB pools using in vivo spectroscopy, western blotting and Fluorescence Recovery After Photobleaching (FRAP) technique, respectively.

Conclusions/Significance

The newly developed model predicts that the phyB-containing nuclear bodies (NBs) (i) serve as storage sites for phyB and (ii) control prolonged dark reversion kinetics as well as partial reversibility of phyB Pfr in extended darkness. The predictive power of this mathematical model is further validated by the fact that we are able to formalize a basic photobiological observation, namely that in light-grown seedlings hypocotyl length depends on the total amount of phyB. In addition, we demonstrate that our theoretical predictions are in excellent agreement with quantitative data concerning phyB levels and the corresponding hypocotyl lengths. Hence, we conclude that the integrative model suggested in this study captures the main features of phyB-mediated photomorphogenesis in Arabidopsis.

Plants use light as a source of information via a suite of photomorphogenic photoreceptors to optimize growth in response to their light environment. Growth-promoting hormones such as brassinosteroids also can modulate many of these responses. BAS1 and SOB7 are brassinosteroid-catabolizing P450s in Arabidopsis thaliana that synergistically/redundantly modulate photomorphogenic traits such as flowering time. The role of BAS1 and SOB7 in photomorphogenesis has been investigated by studying null-mutant genetic interactions with the photoreceptors phyA, phyB, and cry1 with regard to seed germination and flowering time. The removal of BAS1 and/or SOB7 rescued the low germination rate of the phyA-211 phyB-9 double-null mutant. With regard to floral induction, bas1-2 and sob7-1 showed a complex set of genetic interactions with photoreceptor-null mutants. Histochemical analysis of transgenic plants harboring BAS1:BAS1-GUS and SOB7:SOB7-GUS translational fusions under the control of their endogenous promoters revealed overlapping and distinct expression patterns. BAS1’s expression in the shoot apex increases during the phase transition from short-to-long-day growth conditions and requires phyB in red light. In summary, BAS1 and SOB7 displayed both simple and complex genetic interactions with the phytochromes in a plant-stage specific manner.

Background

Over the past decades, extensive comparative mapping research has been performed in the plant family Solanaceae. The recent identification of a large set of single-copy conserved orthologous (COSII) markers has greatly accelerated comparative mapping studies among major solanaceous species including tomato, potato, eggplant, pepper and diploid Nicotiana species (as well as tetraploid tobacco). The large amount of comparative data now available for these species provides the opportunity to describe the overall patterns of chromosomal evolution in this important plant family. The results of this investigation are described herein.

Results

We combined data from multiple COSII studies, and other comparative mapping studies performed in tomato, potato, eggplant, pepper and diploid Nicotiana species, to deduce the features and outcomes of chromosomal evolution in the Solanaceae over the past 30 million years. This includes estimating the rates and timing of chromosomal changes (inversions and translocations) as well as deducing the age of ancestral progenitor species and predicting their genome configurations.

Conclusions

The Solanaceae has experienced chromosomal changes at a modest rate compared with other families and the rates are likely conserved across different lineages of the family. Chromosomal inversions occur at a consistently higher rate than do translocations. Further, we find evidences for non-random positioning of the chromosomal rearrangement breakpoints. This finding is consistent with the similar finding in mammals, where hot spots for chromosomal breakages have apparently played a significant role in shaping genome evolution. Finally, by utilizing multiple genome comparisons we were able to reconstruct the most likely genome configuration for a number of now-extinct progenitor species that gave rise to the extant solanaceous species used in this research. The results from this study provide the first broad overview of chromosomal evolution in the family Solanaceae, and one of the most detailed thus far for any family of plants.

The bHLH transcription factor, PHYTOCHROME INTERACTING FACTOR 3 (PIF3), interacts specifically with the photoactivated, Pfr, form of Arabidopsis phytochrome B (phyB). This interaction induces PIF3 phosphorylation and degradation in vivo and modulates phyB-mediated seedling deetiolation in response to red light. To identify missense mutations in the phyB N-terminal domain that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen individual mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to also disrupt light-induced binding of phyB to PIF3 in in vitro co-immunoprecipitation assays. These phyB missense mutants fall into two general classes: Class I (eleven mutants) containing those defective in light signal perception, due to aberrant chromophore attachment or photoconversion, and Class II (four mutants) containing those normal in signal perception, but defective in the capacity to transduce this signal to PIF3. By generating a homology model for the three-dimensional structure of the Arabidopsis phyB chromophore-binding region, based on the crystal structure of Deinococcus radiodurans phytochrome, we predict that three of the four Class II mutated phyB residues are solvent exposed in a cleft between the presumptive PAS and GAF domains. This deduction suggests that these residues could be directly required for the physical interaction of phyB with PIF3. Because these three residues are also necessary for phyB-imposed inhibition of hypocotyl elongation in response to red light, they are functionally necessary for signal transfer from photoactivated phyB, not only to PIF3 and other related bHLH transcription factors tested here, but also to other downstream signaling components involved in regulating seedling deetiolation.

Author Summary

Plants monitor their environment for informational light signals that are used to direct adaptive morphogenic responses. The phytochrome (phy) family of photoreceptors are central to this process. Upon photoperception, phy molecules rapidly translocate to the nucleus where they interact with basic helix-loop-helix transcription factors, termed PIFs (phy-Interacting Factors), and induce gene-expression changes that control morphogenic responses. The molecular determinants in the phy protein responsible for direct intermolecular signal transfer from the activated photoreceptor to transduction partners are undefined. Using random mutagenesis of Arabidopsis phyB, coupled with a reverse-hybrid protein-interaction screen, we identified missense mutations in the N-terminal domain that abrogate the binding of the photoreceptor molecule to PIF3. A subset of these mutated phyB molecules retain the capacity for light-signal perception but are defective in the capacity to transduce that signal to PIF3 and other related PIFs. The mutated residues in these molecules are predicted to cluster at the surface of the protein in a structure termed the “light-sensing knot.” These residues are necessary for phyB-regulated growth in the living plant, establishing that the protein region identified appears to function as a component of the molecular interface responsible for direct signal transfer to transduction partners in the cell.

Background

Phytochromes are a family of red/far-red photoreceptors that regulate a number of important developmental traits in cotton (Gossypium spp.), including plant architecture, fiber development, and photoperiodic flowering. Little is known about the composition and evolution of the phytochrome gene family in diploid (G. herbaceum, G. raimondii) or allotetraploid (G. hirsutum, G. barbadense) cotton species. The objective of this study was to obtain a preliminary inventory and molecular-evolutionary characterization of the phytochrome gene family in cotton.

Results

We used comparative sequence resources to design low-degeneracy PCR primers that amplify genomic sequence tags (GSTs) for members of the PHYA, PHYB/D, PHYC and PHYE gene sub-families from A- and D-genome diploid and AD-genome allotetraploid Gossypium species. We identified two paralogous PHYA genes (designated PHYA1 and PHYA2) in diploid cottons, the result of a Malvaceae-specific PHYA gene duplication that occurred approximately 14 million years ago (MYA), before the divergence of the A- and D-genome ancestors. We identified a single gene copy of PHYB, PHYC, and PHYE in diploid cottons. The allotetraploid genomes have largely retained the complete gene complements inherited from both of the diploid genome ancestors, with at least four PHYA genes and two genes encoding PHYB, PHYC and PHYE in the AD-genomes. We did not identify a PHYD gene in any cotton genomes examined.

Conclusions

Detailed sequence analysis suggests that phytochrome genes retained after duplication by segmental duplication and allopolyploidy appear to be evolving independently under a birth-and-death-process with strong purifying selection. Our study provides a preliminary phytochrome gene inventory that is necessary and sufficient for further characterization of the biological functions of each of the cotton phytochrome genes, and for the development of 'candidate gene' markers that are potentially useful for cotton improvement via modern marker-assisted selection strategies.

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.

Electronic supplementary material

The online version of this article (doi:10.1007/s11103-013-0029-0) contains supplementary material, which is available to authorized users.

Phytochrome B (phyB), a major photoreceptor in plants, interacts with transcription factors to regulate gene expression and induce various light responses. Recently, we identified an SR-like splicing factor, RRC1 (reduced red-light responses in cry1cry2 background 1), as a novel component of phyB signaling in Arabidopsis. RRC1 has a C-terminal arginine/serine-rich (RS) domain that is generally important for the regulation of alternative splicing. Whereas rrc1 hypomorphic mutant alleles produce truncated RRC1 proteins that lack the C-terminal region, including the RS domain, and exhibit splicing defects and reduced phyB signaling, the rrc1-4 null allele additionally displays pleiotropic developmental abnormalities with more severe splicing defects. Here, we show that transgenic Arabidopsis plants that express truncated RRC1 lacking the RS domain in the rrc1-4 null allele background exhibited the same phenotype as the hypomorphic alleles. Hence, we conclude that deletion of the RS domain of RRC1 reduces phyB signaling, probably due to aberrant regulation of alternative splicing of target genes.

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651–amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, β-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.

Author Summary

Adapting to the light environment, plants have evolved several photoreceptors, of which the phytochromes are specialized in perceiving the red and far-red light region of the spectrum. Although phytochrome was first discovered in plants, the phytochrome species are present in several organisms, including bacteria. The mechanisms by which phytochromes transduce light signals to downstream components are most well studied in plants. Upon light activation, phytochromes translocate from the cytoplasm into nucleus and regulate the gene expression network through interaction with nuclear transcription factors. The phytochrome molecule can be divided into two major domains: the N-terminal moiety, which is responsible for the light perception, and the C-terminal moiety. Although the C-terminal moiety was though to be involved in signal transduction, it has recently been shown that the N-terminal moiety has a role not only in the light perception, but also in light signal transfer to the downstream network. However, no signaling motifs have been found in the N-terminal moiety. In this study, we analyzed intragenic mutations derived from a genetic screen and found a cluster of residues necessary for signal transduction in a small region neighboring the light-sensing chromophore moiety on the three-dimensional structure. This is an important step towards understanding how a major plant photoreceptor, phytochrome, intramolecularly processes the light signal to trigger diverse physiological responses.

In flowering plants, RNA editing is a posttranscriptional process that converts specific C to U in organelle mRNAs. Nicotiana tabacum is an allotetraploid species derived from the progenitors of Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana species have been used as a model for understanding the mechanism and evolution of RNA editing in plastids. In Nicotiana species, the ndhD-1 site is edited to create the translational initiation codon of ndhD that encodes a subunit of the NAD(P)H dehydrogenease (NDH) complex. An analysis of this RNA editing revealed that editing efficiency in N. tomentosiformis is lower (15%) than that in N. tabacum (42%) and N. sylvestris (37%). However, this level of editing is sufficient for accumulating the NDH complex and its activity. The heterogous complementation of Arabidopsis crr4-3 mutant, in which RNA editing of ndhD-1 is completely impaired, with CRR4 orthologous genes derived from Nicotiana species suggested that the reduction in editing efficiency in N. tomentosiformis is caused by amino acid variations accumulating in CRR4.

Dominant gain-of-function alleles of Arabidopsis phytochrome B were recently shown to confer light-independent, constitutive photomorphogenic (cop) phenotypes to transgenic plants (Su and Lagarias, 2007). In the present study, comparative transcription profiling experiments were performed to assess whether the pattern of gene expression regulated by these alleles accurately reflects the process of photomorphogenesis in wild-type Arabidopsis. Whole-genome transcription profiles of dark-grown phyAphyB seedlings expressing the Y276H mutant of phyB (YHB) revealed that YHB reprograms about 13% of the Arabidopsis transcriptome in a light-independent manner. The YHB-regulated transcriptome proved qualitatively similar to but quantitatively greater than those of wild-type seedlings grown under 15 or 50 μmol m−2 m−1 continuous red light (Rc). Among the 2977 genes statistically significant two-fold (SSTF) regulated by YHB in the absence of light include those encoding components of the photosynthetic apparatus, tetrapyrrole/pigment biosynthetic pathways, and early light-responsive signaling factors. Approximately 80% of genes SSTF regulated by Rc were also YHB-regulated. Expression of a notable subset of 346 YHB-regulated genes proved to be strongly attenuated by Rc, indicating compensating regulation by phyC-E and/or other Rc-dependent processes. Since the majority of these 346 genes are regulated by the circadian clock, these results suggest that phyA- and phyB-independent light signaling pathway(s) strongly influence clock output. Together with the unique plastid morphology of dark-grown YHB seedlings, these analyses indicate that the YHB mutant induces constitutive photomorphogenesis via faithful reconstruction of phyB signaling pathways in a light-independent fashion.

Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859–868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.

Background

Regulation of gene expression at the level of transcription is a major control point in many biological processes. Transcription factors (TFs) can activate and/or repress the transcriptional rate of target genes and vascular plant genomes devote approximately 7% of their coding capacity to TFs. Global analysis of TFs has only been performed for three complete higher plant genomes – Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa) and rice (Oryza sativa). Presently, no large-scale analysis of TFs has been made from a member of the Solanaceae, one of the most important families of vascular plants. To fill this void, we have analysed tobacco (Nicotiana tabacum) TFs using a dataset of 1,159,022 gene-space sequence reads (GSRs) obtained by methylation filtering of the tobacco genome. An analytical pipeline was developed to isolate TF sequences from the GSR data set. This involved multiple (typically 10–15) independent searches with different versions of the TF family-defining domain(s) (normally the DNA-binding domain) followed by assembly into contigs and verification. Our analysis revealed that tobacco contains a minimum of 2,513 TFs representing all of the 64 well-characterised plant TF families. The number of TFs in tobacco is higher than previously reported for Arabidopsis and rice.

Results

TOBFAC: the database of tobacco transcription factors, is an integrative database that provides a portal to sequence and phylogeny data for the identified TFs, together with a large quantity of other data concerning TFs in tobacco. The database contains an individual page dedicated to each of the 64 TF families. These contain background information, domain architecture via Pfam links, a list of all sequences and an assessment of the minimum number of TFs in this family in tobacco. Downloadable phylogenetic trees of the major families are provided along with detailed information on the bioinformatic pipeline that was used to find all family members. TOBFAC also contains EST data, a list of published tobacco TFs and a list of papers concerning tobacco TFs. The sequences and annotation data are stored in relational tables using a PostgrelSQL relational database management system. The data processing and analysis pipelines used the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The computationally intensive data processing and analysis pipelines were run on an Apple XServe cluster with more than 20 nodes.

Conclusion

TOBFAC is an expandable knowledgebase of tobacco TFs with data currently available for over 2,513 TFs from 64 gene families. TOBFAC integrates available sequence information, phylogenetic analysis, and EST data with published reports on tobacco TF function. The database provides a major resource for the study of gene expression in tobacco and the Solanaceae and helps to fill a current gap in studies of TF families across the plant kingdom. TOBFAC is publicly accessible at .

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.

The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.

Summary

Plants respond to a reduction in the red:far-red ratio (R:FR) of light caused by the proximity of other plants by initiating morphological changes that improve light capture. In Arabidopsis, this response (the shade avoidance syndrome, SAS) is controlled by the phytochromes (particularly phyB) and dependent on the TAA1 pathway of auxin biosynthesis. However, when grown in real canopies, we found that phyB mutants and mutants deficient in TAAI (sav3) still display robust SAS responses to increased planting density and leaf shading. The SAS morphology (leaf hyponasty and reduced lamina:petiole ratio) could be phenocopied by exposing plants to blue (B) light attenuation. These responses to B light attenuation required the UV-A/blue light photoreceptor cry1. Moreover, they were mediated through mechanisms that showed only limited overlap with the pathways recruited by phyB inactivation. In particular, pathways for polar auxin transport, auxin biosynthesis and gibberellin signaling that are involved in SAS responses to low R:FR were not required for the SAS responses to B light depletion. By contrast, brassinosteroid response appeared to be required for full expression of the SAS phenotype under low B light. The phyB and cry1 inactivation pathways appeared to converge in their requirement for the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and 5) to elicit the SAS phenotype. Our results suggest that B light is an important control of SAS responses, and that PIF4 and PIF5 are critical hubs for a diverse array of signaling routes that control plant architecture in canopies.

Plants respond to a reduction in the red/far-red ratio (R:FR) of light, caused by the proximity of other plants, by initiating morphological changes that improve light capture. In Arabidopsis, this response (shade avoidance syndrome, SAS) is controlled by phytochromes (particularly phyB), and is dependent on the TAA1 pathway of auxin biosynthesis. However, when grown in real canopies, we found that phyB mutants and mutants deficient in TAAI (sav3) still display robust SAS responses to increased planting density and leaf shading. The SAS morphology (leaf hyponasty and reduced lamina/petiole ratio) could be phenocopied by exposing plants to blue light attenuation. These responses to blue light attenuation required the UV-A/blue light photoreceptor cry1. Moreover, they were mediated through mechanisms that showed only limited overlap with the pathways recruited by phyB inactivation. In particular, pathways for polar auxin transport, auxin biosynthesis and gibberellin signaling that are involved in SAS responses to low R:FR were not required for the SAS responses to blue light depletion. By contrast, the brassinosteroid response appeared to be required for the full expression of the SAS phenotype under low blue light. The phyB and cry1 inactivation pathways appeared to converge in their requirement for the basic/helix-loop-helix (bHLH) transcription factors PHYTOCHROME INTERACTING FACTORs 4 and 5 (PIF4 and PIF5) to elicit the SAS phenotype. Our results suggest that blue light is an important control of SAS responses, and that PIF4 and PIF5 are critical hubs for a diverse array of signaling routes that control plant architecture in canopies.