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Plant terrestrialization brought forth the land plants (embryophytes). Embryophytes account for most of the biomass on land and evolved from streptophyte algae in a singular event. Recent advances have unraveled the first full genomes of the closest algal relatives of land plants; among the first such species wasMesotaenium endlicherianum. Here, we used fine-combed RNAseq in tandem with photophysiological assessment onMesotaeniumexposed to a continuous range of temperature and light cues. Our data establish a grid of 42 different conditions, resulting in 128 transcriptomes and ~1.5 Tbp (~9.9 billion reads) of data to study combinatory effects of stress response using clustering along gradients. We describe major hubs in genetic networks underpinning stress response and acclimation in the molecular physiology ofMesotaenium. Our data suggest that lipid droplet formation, plastid and cell wall-derived signals denominate molecular programs since more than 600 million years of streptophyte evolution—before plants made their first steps on land.

Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme β-galactosidase (β-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) is an inexpensive and sensitive substrate of β-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5'-dibromo-4,4'-dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent yeast cells and transformation Alternate Protocol 1: In-house preparation of yeast competent cells for use in lithium acetate (LiAc)-mediated yeast transformation Support Protocol: Long-term storage and revival of frozen yeast strain stocks Basic Protocol 2: Measuring β-galactosidase activity via the quantitative X-gal assay Alternate Protocol 2: Quantification of interaction strength using liquid ONPG assay.

Arabidopsis (Arabidopsis thaliana) CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and members of the SUPPRESSOR OF PHYTOCHROMEA-105 (SPA) protein family form an E3 ubiquitin ligase that suppresses light signaling in darkness by polyubiquitinating positive regulators of the light response. COP1/SPA is inactivated by light to allow photomorphogenesis to proceed. Mechanisms of inactivation include light-induced degradation of SPA1 and, in particular, SPA2, corresponding to a particularly efficient inactivation of COP1/SPA2 by light. Here, we show that SPA3 and SPA4 proteins are stable in the light, indicating that light-induced destabilization is specific to SPA1 and SPA2, possibly related to the predominant function of SPA1 and SPA2 in dark-grown etiolating seedlings. SPA2 degradation involves cullin and the COP10-DEETIOLATED-DAMAGED-DNA BINDING PROTEIN (DDB1) CDD complex, besides COP1. Consistent with this finding, light-induced SPA2 degradation required the DDB1-interacting Trp-Asp (WD)-repeat domain of SPA2. Deletion of the N-terminus of SPA2 containing the kinase domain led to strong stabilization of SPA2 in darkness and fully abolished light-induced degradation of SPA2. This prevented seedling de-etiolation even in very strong far-red and blue light and reduced de-etiolation in red light, indicating destabilization of SPA2 through its N-terminal domain is essential for light response. SPA2 is exclusively destabilized by phytochrome A in far-red and blue light. However, deletion of the N-terminal domain of SPA2 did not abolish SPA2-phytochrome A interaction in yeast nor in vivo. Our domain mapping suggests there are two SPA2-phytochrome A interacting domains, the N-terminal domain and the WD-repeat domain. Conferring a light-induced SPA2-phyA interaction only via the WD-repeat domain may thus not lead to COP1/SPA2 inactivation.

In plants, the cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40-binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays and in planta Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

The biosynthesis of defensive secondary metabolites, such as glucosinolates (GSLs), is a costly process, which requires nutrients, ATP, and reduction equivalents, and, therefore, needs well-orchestrated machinery while coordinating defense and growth. We discovered that the key repressor of light signaling, the CONSTITUTIVE PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYTOCHROME A-105 (COP1/SPA) complex, is a crucial component of GSL biosynthesis regulation. Various mutants in this COP1/SPA complex exhibited a strongly reduced level of GSL and a low expression of jasmonate (JA)-dependent genes. Furthermore, cop1, which is known to accumulate DELLA proteins in the dark, shows reduced gibberellin (GA) and JA signaling, thereby phenocopying other DELLA-accumulating mutants. This phenotype can be complemented by a dominant gain-of-function allele of MYC3 and by crossing with a mutant having low DELLA protein levels. Hence, SPA1 interacts with DELLA proteins in a yeast two-hybrid screen, whereas high levels of DELLA inhibit MYC function and suppress JA signaling. DELLA accumulation leads to reduced synthesis of GSL and inhibited growth. Thus, the COP1/SPA-mediated degradation of DELLA not only affects growth but also regulates the biosynthesis of GSLs.

The Arabidopsis COP1/SPA complex acts as a cullin4-based E3 ubiquitin ligase to suppress photomorphogenesis in darkness. It is a tetrameric complex of two COP1 and two SPA proteins. Both COP1 and SPA are essential for the activity of this complex, and they both contain a C-terminal WD-repeat domain responsible for substrate recruitment and binding of DDB1. Here, we used a WD domain swap-approach to address the cooperativity of COP1 and SPA proteins. We found that expression of a chimeric COP1 carrying the WD-repeat domain of SPA1 mostly complemented the cop1-4 mutant phenotype in darkness, indicating that the WD-repeat of SPA1 can replace the WD of COP1. In the light, SPA1-WD partially substituted for COP1-WD. In contrast, expression of a chimeric SPA1 protein carrying the WD-repeat of COP1 did not rescue the spa mutant phenotype. Together, our findings demonstrate that a SPA1-type WD-repeat is essential for COP1/SPA activity, while a COP1-type WD is in part dispensible. Moreover, a complex with four SPA1-WDs is more active than a complex with only two SPA1-WDs. A homology model of SPA1-WD based on the crystal structure of COP1-WD uncovered two insertions and several amino acid substitutions at the predicted substrate-binding pocket of SPA1-WD. This article is protected by copyright. All rights reserved.

The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage. To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb. Light-grown PpspaAB double mutants exhibit smaller gametophores than the wild-type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light-regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark-grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb. The data suggest that COP1/SPA-mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness.

Phytochromes are photoreceptors regulating growth and development in plants. Using the model plant Arabidopsis, we identified a novel signalling pathway downstream of the far-red light-sensing phytochrome, phyA, that depends on the highly conserved CCR4-NOT complex. CCR4-NOT is integral to RNA metabolism in yeast and animals, but its function in plants is largely unknown. NOT9B, an Arabidopsis homologue of human CNOT9, is a component of the CCR4-NOT complex, and acts as negative regulator of phyA-specific light signalling when bound to NOT1, the scaffold protein of the complex. Light-activated phyA interacts with and displaces NOT9B from NOT1, suggesting a potential mechanism for light signalling through CCR4-NOT. ARGONAUTE 1 and proteins involved in splicing associate with NOT9B and we show that NOT9B is required for specific phyA-dependent alternative splicing events. Furthermore, association with nuclear localised ARGONAUTE 1 raises the possibility that NOT9B and CCR4-NOT are involved in phyA-modulated gene expression., eLife digest Place a seedling on a windowsill, and soon you will notice the fragile stem bending towards the glass to soak in the sun and optimize its growth. Plants can ‘sense’ light thanks to specialized photoreceptor molecules: for instance, the phytochrome A is responsible for detecting weak and ‘far-red’ light from the very edge of the visible spectrum. Once the phytochrome has been activated, this message is relayed to the rest of the plant through an intricate process that requires other molecules. The CCR4-NOT protein complex is vital for all plants, animals and fungi, suggesting that it was already present in early life forms. Here, Schwenk et al. examine whether CCR4-NOT could have acquired a new role in plants to help them respond to far-red light. Scanning the genetic information of the plant model Arabidopsis thaliana revealed that the gene encoding the NOT9 subunit of CCR4-NOT had been duplicated in plants during evolution. NOT9B, the protein that the new copy codes for, has a docking site that can attach to both phytochrome A and CCR4-NOT. When NOT9B binds phytochrome A, it is released from the CCR4-NOT complex: this could trigger a cascade of reactions that ultimately changes how A. thaliana responds to far-red light. Plants that had not enough or too much NOT9B were respectively more or less responsive to that type of light, showing that the duplication of the gene coding for this subunit had helped plants respond to certain types of light. The findings by Schwenk et al. illustrate how existing structures can be repurposed during evolution to carry new roles. They also provide a deeper understanding of how plants optimize their growth, a useful piece of information in a world where most people rely on crops as their main source of nutrients.

Photomorphogenesis is repressed in the dark mainly by an E3 ubiquitin ligase complex comprising CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and four homologous proteins called SUPPRESSOR OF PHYA-105 (SPA1-SPA4) in Arabidopsis. This complex induces the ubiquitination and subsequent degradation of positively acting transcription factors (e.g., HY5, HFR1, PAP1 and others) in the dark to repress photomorphogenesis. Genomic evidence showed a large number of genes regulated by COP1 in the dark, of which many are direct targets of HY5. However, the genomic basis for the constitute photomorphogenic phenotype of spaQ remains unknown. Here, we show that >7200 genes are differentially expressed in the spaQ background compared to wild-type in the dark. Comparison of the RNA Sequencing (RNA-Seq) data between cop1 and spaQ revealed a large overlapping set of genes regulated by the COP1-SPA complex. In addition, many of the genes coordinately regulated by the COP1-SPA complex are also regulated by HY5 directly and indirectly. Taken together, our data reveal that SPA proteins repress photomorphogenesis by controlling gene expression in concert with COP1, likely through regulating the abundance of downstream transcription factors in light signaling pathways. Moreover, SPA proteins may function both in a COP1-dependent and –independent manner in regulating many biological processes and developmental pathways in Arabidopsis.Summary statementComparison of transcriptome analyses between cop1 and spaQ mutants reveal overlapping pathways regulated by COP1 and SPAs.

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these path- ways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homo- logue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, far- red and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light sig- nalling during early seedling development, thereby preventing an exaggerated response to light.

In plants, the cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40–binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays and in planta. Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

The ambient light environment controls many aspects of plant development throughout a plant’s life cycle. Such complex control is achieved because a key repressor of light signaling, the Arabidopsis (Arabidopsis thaliana) COP1/SPA E3 ubiquitin ligase causes the degradation of multiple regulators of endogenous developmental pathways. This includes the CONSTANS (CO) transcription factor that is responsible for photoperiodic control of flowering time. There are 16 CO-like proteins whose functions are only partly understood. Here, we show that 14 CO-like (COL) proteins bind CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYTOCHROME A-105 (SPA)1 in vitro. We subsequently focused on COL12 and show that COL12 binds COP1 and SPA proteins in vivo. The COL12 protein is degraded in darkness in a COP1-dependent fashion, indicating that COL12 is a substrate of the COP1/SPA ubiquitin ligase. Overexpression of COL12 causes late flowering specifically in long day conditions by decreasing the expression of FLOWERING LOCUS T. This phenotype is genetically dependent on CO. Consistent with this finding, COL12 physically interacts with CO in vivo, suggesting that COL12 represses flowering by inhibiting CO protein function. We show that COL12 overexpression did not alter CO protein stability. It is therefore likely that COL12 represses the activity of CO rather than CO levels. Overexpression of COL12 also affects plant architecture by increasing the number of rosette branches and reducing inflorescence height. These phenotypes are CO independent. Hence, we suggest that COL12 affects plant development through CO-dependent and CO-independent mechanisms.

Light is a critical signal to integrate plant growth and development with the environment. Downstream of photoreceptors, the E3 ubiquitin ligase COP1/SPA is a key repressor of photomorphogenesis which targets many positive regulators of light signaling, mainly transcription factors, for degradation in darkness. In light-grown plants COP1/SPA activity is repressed, allowing light responses to occur. This review provides an overview on our current knowledge on COP1/SPA repressor function, focusing in particular on the roles of the respective protein domains and the mechanisms of light-induced inactivation of COP1/SPA. Moreover, we summarize how COP1 activity is regulated by other interacting proteins, such as a SUMO E3 ligase and Phytochrome-Interacting Factors (PIFs), as well as by hormones. At last, several novel functions of COP1 that were recently revealed are included.

The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage. To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb. Light-grown PpspaAB double mutants exhibit smaller gametophores than the wild-type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light-regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark-grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb. The data suggest that COP1/SPA-mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness.

Plants are continuously exposed to a myriad of stresses, which lead to the formation of secondary metabolites including flavonoids. Studies suggest that low temperature exposure leads to enhanced flavonoid accumulation in Arabidopsis thaliana. In addition, flavonoid biosynthesis is regulated by light through various regulatory factors. Therefore, plants may possess the capability to integrate light and low temperature signals for survival under freezing conditions. However, the detailed molecular mechanism and the regulatory factors associated with light- and low temperature- responsive flavonoid biosynthesis remain largely unknown. Here, we report a strict requirement for light for the low temperature-enhanced flavonol biosynthesis. Low temperature-induced expression of biosynthetic genes as well as flavonol accumulation was hampered in ELONGATED HYPOCOTYL (hy5) and myb11myb111myb12 triple mutants as compared with the wild type in Arabidopsis. Overexpression of AtHY5 in the hy5 mutant restored induction of gene expression and flavonol accumulation in response to low temperature in light. Metabolite and gene expression analysis also suggests a negative role for CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in accumulation of flavonols in response to low temperature. Overexpression of AtMYB12 enhanced accumulation of flavonols under low temperature in a light-dependent manner. Together, our analysis suggests the requirement for HY5 and flavonol-specific MYB regulatory factors for low temperature-induced flavonol synthesis.

The Arabidopsis (Arabidopsis thaliana) COP1/SPA ubiquitin ligase is a central repressor that suppresses light signaling in darkness by targeting positive regulators of the light response, mainly transcription factors, for degradation. Light inactivates COP1/SPA, in part by excluding COP1 from the nucleus. SPA proteins are essential cofactors of COP1, but their exact role in the COP1/SPA complex is thus far unknown. To unravel a potential role of SPA proteins in COP1 nucleocytoplasmic partitioning, we monitored the subcellular localization of COP1 in a spa1234 quadruple mutant (spaQn). We analyzed a YFP-COP1-expressing transgenic line and endogenous COP1 after subcellular fractionation. In dark-grown seedlings, both YFP-COP1 and endogenous COP1 accumulated in the nucleus in the absence and presence of SPA proteins, indicating that SPA proteins are not required for nuclear localization of COP1 in darkness. In contrast, in white light-grown seedlings, spaQn mutants failed to relocalize COP1 from the nucleus to the cytoplasm. Hence, SPA proteins are necessary for the light-controlled change in COP1 subcellular localization. We conclude that SPA proteins have a dual role: (1) they are required for light-responsiveness of COP1 subcellular localization, and (2) they promote COP1 activity in darkness in a fashion that is independent of the nuclear import/nuclear retention of COP1.

Stomatal development is tightly regulated through internal and external factors that are integrated by a complex signalling network. Light represents an external factor that strongly promotes stomata formation. Here, we show that auxin-resistant aux/iaa mutants, e.g. axr3-1, exhibit a de-repression of stomata differentiation in dark-grown seedlings. The higher stomatal index in dark-grown axr3-1 mutants when compared with the wild type is due to increased cell division in the stomatal lineage. Excessive stomata in dark-grown seedlings were also observed in mutants defective in auxin biosynthesis or auxin perception and in seedlings treated with the polar auxin transport inhibitor NPA. Consistent with these findings, exogenous auxin repressed stomata formation in light-grown seedlings. Taken together, these results indicate that auxin is a negative regulator of stomatal development in dark-grown seedlings. Epistasis analysis revealed that axr3-1 acts genetically upstream of the bHLH transcription factors SPCH, MUTE and FAMA, as well as the YDA MAP kinase cascade, but in parallel with the repressor of photomorphogenesis COP1 and the receptor-like protein TMM. The effect of exogenous auxin required the ER family of leucine-rich repeat receptor-like kinases, suggesting that auxin acts at least in part through the ER family. Expression of axr3-1 in the stomatal lineage was insufficient to alter the stomatal index, implying that cell-cell communication is necessary to mediate the effect of auxin. In summary, our results show that auxin signalling contributes to the suppression of stomatal differentiation observed in dark-grown seedlings.

The Arabidopsis CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex is a key repressor of light signaling that inhibits light responses in darkness. It acts as an E3 ubiquitin ligase, which ubiquitinates positively acting light-signaling intermediates, mainly transcription factors, thereby targeting them for proteolytic degradation by the 26S proteasome. In the light, photoreceptors directly interact with the COP1/SPA complex, leading to its inactivation, which subsequently allows the target transcription factors to accumulate and initiate vast reprogramming of gene expression (Huang et al., 2014).

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post-germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub-nuclear accumulation of COP1. The MID protein is not degraded in a COP1-dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1-dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1-dependent development with the regulation of endoreduplication.

International audience; The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This DN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis.

Background Plants have evolved complex mechanisms to adapt growth and development to the light environment. The COP1/SPA complex is a key repressor of photomorphogenesis in dark-grown Arabidopsis plants and acts as an E3 ubiquitin ligase to ubiquitinate transcription factors involved in the light response. In the light, COP1/SPA activity is inhibited by photoreceptors, thereby allowing accumulation of these transcription factors and a subsequent light response. Previous results have shown that the four members of the SPA family exhibit partially divergent functions. In particular, SPA1 and SPA2 strongly differ in their responsiveness to light, while they have indistinguishable activities in darkness. The much higher light-responsiveness of SPA2 is partially explained by the much stronger light-induced degradation of SPA2 when compared to SPA1. Here, we have conducted SPA1/SPA2 domain swap experiments to identify the protein domain(s) responsible for the functional divergence between SPA1 and SPA2. Results We have individually swapped the three domains between SPA1 and SPA2 - the N-terminal kinase-like domain, the coiled-coil domain and the WD-repeat domain - and expressed them in spa mutant Arabidopsis plants. The phenotypes of transgenic seedlings show that the respective N-terminal kinase-like domain is primarily responsible for the respective light-responsiveness of SPA1 and SPA2. Furthermore, the most divergent part of the N-terminal domain was sufficient to confer a SPA1- or SPA2-like activity to the respective SPA protein. The stronger light-induced degradation of SPA2 when compared to SPA1 was also primarily conferred by the SPA2 N-terminal domain. At last, the different affinities of SPA1 and SPA2 for cryptochrome 2 are defined by the N-terminal domain of the respective SPA protein. In contrast, both SPA1 and SPA2 similarly interacted with COP1 in light-grown seedlings. Conclusions Our results show that the distinct activities and protein stabilities of SPA1 and SPA2 in light-grown seedlings are primarily encoded by their N-terminal kinase-like domains. Similarly, the different affinities of SPA1 and SPA2 for cry2 are explained by their respective N-terminal domain. Hence, after a duplication event during evolution, the N-terminal domains of SPA1 and SPA2 underwent subfunctionalization, possibly to allow optimal adaptation of growth and development to a changing light environment. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0854-9) contains supplementary material, which is available to authorized users.

Plants sense vegetative shade as a reduction in the ratio of red light to far-red light (R:FR). Arabidopsis (Arabidopsis thaliana) responds to a reduced R:FR with increased elongation of the hypocotyl and the leaf petioles as well as with an acceleration of flowering time. The repressor of light signaling, CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), has been shown previously to be essential for the shade-avoidance response in seedlings. Here, we have investigated the roles of COP1 and the COP1-interacting SUPPRESSOR OF PHYA-105 (SPA) proteins in seedling and adult facets of the shade-avoidance response. We show that COP1 and the four SPA genes are essential for hypocotyl and leaf petiole elongation in response to low R:FR, in a fashion that involves the COP1/SPA ubiquitination target LONG HYPOCOTYL IN FR LIGHT1 but not ELONGATED HYPOCOTYL5. In contrast, the acceleration of flowering in response to a low R:FR was normal in cop1 and spa mutants, thus demonstrating that the COP1/SPA complex is only required for elongation responses to vegetative shade and not for shade-induced early flowering. We further show that spa mutant seedlings fail to exhibit an increase in the transcript levels of the auxin biosynthesis genes YUCCA2 (YUC2), YUC8, and YUC9 in response to low R:FR, suggesting that an increase in auxin biosynthesis in vegetative shade requires SPA function. Consistent with this finding, expression of the auxin-response marker gene DR5::GUS did not increase in spa mutant seedlings exposed to low R:FR. We propose that COP1/SPA activity, via LONG HYPOCOTYL IN FR LIGHT1, is required for shade-induced modulation of the auxin biosynthesis pathway and thereby enhances cell elongation in low R:FR.

During analysis of an Arabidopsis thaliana line possessing a Phytochrome A epiallele (phyA’), a partial Phytochrome B-deficient phenotype was observed, consisting of elongated hypocotyls in seedlings grown under constant white light or red light (660 nm). The observed hypocotyls were twice the length (8 mm) of wild-type (4 mm), but approximately half the length of a null phyB-9 mutant (14 mm). Several analyses were performed to characterize this apparent partial phyB mutant. Sequencing of the entire exonic region revealed three point mutations that altered codon usage, and one in-frame 12 base pair deletion. Each of the point mutations has been described in other lines that display wild-type phenotype, and therefore their effect is thought to be minimal, if any. The N-terminal deletion of amino acids 9 through 12 (GGGR) is a unique mutation found in this line. This deletion most likely contributes to the phyB mutant phenotype by lowering the binding affinity of the active form of Phytochrome B (Pfr) with Phytochrome Interacting Factor 3 (PIF3).

Summary The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1–SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.

The COP1/SPA complex and DET1 function to suppress photomorphogenesis in dark-grown Arabidopsis seedlings. Additionally, they inhibit flowering under non-inductive short-day conditions. The COP1/SPA complex and DET1, as part of the CDD complex, represent distinct high-molecular-weight complexes in Arabidopsis. Here, we provide genetic evidence that these complexes co-act in regulating plant development. We report the isolation of a spa1 enhancer mutation that represents a novel, very weak allele of det1. This det1 esp1 mutation caused no detectable mutant phenotype in the presence of wild-type SPA1, but showed strongly synergistic genetic interaction with the spa1 mutation in the control of seedling photomorphogenesis, anthocyanin accumulation, plant size as well as flowering time. On the biochemical level, the det1 esp1 spa1 double mutant showed higher HY5 protein levels than either single mutant or the wild type. The genetic interaction of spa1 and det1 mutations was further confirmed in the spa1 det1-1 double mutant which carries a strong allele of det1. Taken together, these results show that SPA1 and DET1 act together to control photomorphogenesis throughout plant development. Hence, this suggests that COP1/SPA complexes and the CDD complex co-act in controlling the protein stability of COP1/SPA target proteins.

Fine tuning of light signaling is crucial to plant development. Following light-triggered nuclear translocation, the photoreceptor phytochrome A (phyA) regulates gene expression under continuous far-red light and is rapidly destabilized upon red light irradiation by E3 ubiquitin ligases, including COP1. Here we provide evidence that the light signaling repressors SPA proteins contribute to COP1-mediated phyA degradation and that a COP1/SPA1 protein complex is tightly associated with phyA ubiquitination activity. Furthermore, a phosphorylated phyA form accumulates in the nucleus and preferentially associates with the COP1/SPA1 complex. In contrast, underphosphorylated phyA predominantly associates with the phyA-signaling intermediates FHY3 and FHY1. However, COP1 associates with underphosphorylated phyA in the absence of FHY3 or FHY1, suggesting that phyA associations with FHY3 and FHY1 protect underphosphorylated phyA from being recognized by the COP1/SPA complex. We propose that light-induced phyA phosphorylation acts as a switch controlling differential interactions of the photoreceptor with signal propagation or attenuation machineries.

The four-member SPA protein family of Arabidopsis acts in concert with the E3 ubiquitin ligase COP1 to suppress photomorphogenesis in dark-grown seedlings. Here, we demonstrate that SPA proteins are, moreover, essential for photoperiodic flowering. Mutations in SPA1 cause phyA-independent early flowering under short day (SD) but not long day (LD) conditions, and this phenotype is enhanced by additional loss of SPA3 and SPA4 function. These spa1 spa3 spa4 triple mutants flower at the same time in LD and SD, indicating that the SPA gene family is essential for the inhibition of flowering under non-inductive SD. Among the four SPA genes, SPA1 is necessary and sufficient for normal photoperiodic flowering. Early flowering of SD-grown spa mutant correlates with strongly increased FT transcript levels, whereas COtranscript levels are not altered. Epistasis analysis demonstrates that both early flowering and FT induction in spa1 mutants is fully dependent on CO. Consistent with this finding, SPA proteins interact physically with CO in vitro and in vivo, suggesting that SPA proteins regulate CO protein function. Domain mapping shows that the SPA1-CO interaction requires the CCT-domain of CO, but is independent of the B-box type Zn fingers of CO. We further show that spa1 spa3 spa4 mutants exhibit strongly increased CO protein levels, which are not caused by a change in COgene expression. Taken together, our results suggest, that SPA proteins regulate photoperiodic flowering by controlling the stability of the floral inducer CO.

The four members of the Suppressor of phyA-105 (SPA) gene family function to inhibit photomorphogenesis in dark- and light-grown seedlings. Additionally, SPA1-SPA4 regulate elongation growth of adult plants. In these processes, SPA2, SPA3 and SPA4 have overlapping but distinct functions. Here, we have further investigated the role of SPA1 which is partially masked by functional redundancy. We show that SPA1 represses not only red, far-red and blue light responses in a PHYA-dependent fashion, but also acts to suppress light signaling in darkness. We demonstrate that deletion-derivatives of SPA1 lacking the complete N-terminus or part of the kinase-like domain retain SPA1 function in light- and dark-grown seedlings, while deletion of the constitutive photomorphogenesis 1 (COP1)-interacting coiled-coil domain eliminates SPA1 activity. This suggests that the coiled-coil domain and the WD-repeat domain of SPA1 are sufficient for SPA1 function. An analysis of spa2 spa3 spa4 triple mutants demonstrates that SPA1, like SPA2, is sufficient for normal etiolation of dark-grown seedlings. In light-grown seedlings and adult plants, in contrast, SPA1 function is divergent from SPA2 function, with SPA1 playing the predominant role. Levels of SPA1, SPA3 and SPA4 transcript are increased by red, far-red and blue light, consistent with a role of these three SPA genes in light-grown seedlings. The abundance of SPA2 mRNA, in contrast, is not altered by light. Taken together, the analysis of SPA transcript levels suggests that differences in SPA gene expression patterns contribute to divergence in SPA1-SPA4 function.

Arabidopsis uses two major classes of photoreceptors to mediate seedling de-etiolation. The cryptochromes (cry1 and cry2) absorb blue/ultraviolet-A light, whereas the phytochromes (phyA-phyE) predominantly regulate responses to red/far-red light. Arabidopsis COP1 represses light signaling by acting as an E3 ubiquitin ligase in the nucleus, and is responsible for targeted degradation of a number of photomorphogenesis-promoting factors, including HY5, LAF1, phyA, and HFR1. Distinct light signaling pathways initiated by multiple photoreceptors (including both phytochromes and cryptochromes) eventually converge on COP1, causing its inactivation and nuclear depletion. Arabidopsis SPA1, which encodes a protein structurally related to COP1, also represses light signaling under various light conditions. In this study, we present genetic evidence supporting that HFR1, which encodes a photomorphogenesis-promoting bHLH transcription factor, acts downstream of SPA1 and is required for different subsets of branch pathways of light signaling controlled by SPA1 under different light conditions. We show that SPA1 physically interacts with HFR1 in a yeast two-hybrid assay and an in vitro co-immunoprecipitation assay. We demonstrate that higher levels of HFR1 protein accumulate in the spa1 mutant background under various light conditions, including far-red, red, blue, and white light, whereas a marginal increase in HFR1 transcript level is only seen in dark- and far-red light-grown spa1-100 mutants. Together, our data suggest that repression of light signaling by Arabidopsis SPA1 likely involves post-translational regulation of HFR1 protein accumulation.

The Arabidopsis thaliana proteins suppressor of phytochrome A-105 1 (SPA1), SPA3, and SPA4 of the four-member SPA1 protein family have been shown to repress photomorphogenesis in light-grown seedlings. Here, we demonstrate that spa quadruple mutant seedlings with defects in SPA1, SPA2, SPA3, and SPA4 undergo strong constitutive photomorphogenesis in the dark. Consistent with this finding, adult spa quadruple mutants are extremely small and dwarfed. These extreme phenotypes are only observed when all SPA genes are mutated, indicating functional redundancy among SPA genes. Differential contributions of individual SPA genes were revealed by analysis of spa double and triple mutant genotypes. SPA1 and SPA2 predominate in dark-grown seedlings, whereas SPA3 and SPA4 prevalently regulate the elongation growth in adult plants. Further analysis of SPA2 function indicated that SPA2 is a potent repressor of photomorphogenesis only in the dark but not in the light. The SPA2 protein is constitutively nuclear localized in planta and can physically interact with the repressor COP1. Epistasis analysis between spa2 and cop1 mutations provides strong genetic support for a biological significance of a COP1-SPA2 interaction in the plant. Taken together, our results have identified a new family of proteins that is essential for suppression of photomorphogenesis in darkness.

Arabidopsis COP1 is a constitutive repressor of photomorphogenesis that interacts with photomorphogenesis-promoting factors such as HY5 to promote their proteasome-mediated degradation. SPA1 is a repressor of phytochrome A-mediated responses to far-red light. Here we report that COP1 acts as part of a large protein complex and interacts with SPA1 in a light-dependent manner. We further demonstrate the E3 ubiquitin ligase activity of COP1 on HY5 in vitro and the alteration of that activity by SPA1. Thus, the COP1-SPA1 interaction defines a critical step in coordinating COP1-mediated ubiquitination and subsequent degradation of HY5 with PHYA signaling.

In Arabidopsis and many other plant species, anthocyanin pigments accumulate only after light exposure and not in darkness. Excess light of very high fluence rates leads to a further, very strong increase in anthocyanin levels. How excess light is sensed is not well understood. Here, we show that mutations in the key repressor of light signaling, the COP1/SPA complex, cause a strong hyperaccumulation of anthocyanins not only under normal light but also under excess, high light conditions. Hence, normal light signaling via COP1/SPA is required to prevent hyperaccumulation of anthocyanins under these high light conditions. However, since cop1 and spa mutants show a similar high-light responsiveness of anthocyanin accumulation as the wild type it remains to be resolved whether COP1/SPA is directly involved in the high-light response itself.

Three recent publications identify the phytohormone auxin as a novel regulator of stomata development and distribution. We discuss how auxin signaling and polar auxin transport blend in with the established signaling network that controls cell division and differentiation within the stomatal cell lineage.

Background: Plants have evolved light sensing mechanisms to optimally adapt their growth and development to the ambient light environment. The COP1/SPA complex is a key negative regulator of light signaling in the well-studied dicot Arabidopsis thaliana. COP1 and members of the four SPA proteins are part of an E3 ubiquitin ligase that acts in darkness to ubiquitinate several transcription factors involved in light responses, thereby targeting them for degradation by the proteasome. While COP1 is also found in humans, SPA proteins appear specific to plants. Here, we have functionally addressed evolutionary conservation of COP1 and SPA orthologs from the moss Physcomitrella, the monocot rice and the dicot Arabidopsis. Results: To this end, we analyzed the activities of COP1- and SPA-like proteins from Physcomitrella patens and rice when expressed in Arabidopsis. Expression of r ice COP1 and Physcomitrella COP1 protein sequences predominantly complemented all phenotypic aspects of the viable, hypomorphic cop1-4 mutant and the null, seedling-lethal cop1-5 mutant of Arabidopsis: rice COP1 fully rescued the constitutive-photomorphogenesis phenotype in darkness and the leaf expansion defect of cop1 mutants, while it partially restored normal photoperiodic flowering in cop1. Physcomitrella COP1 partially restored normal seedling growth and flowering time, while it fully restored normal leaf expansion in the cop1 mutants. In contrast, expression of a SPA ortholog from Physcomitrella (PpSPAb) in Arabidopsis spa mutants did not rescue any facet of the spa mutant phenotype, suggesting that the PpSPAb protein is not functionally conserved or that the Arabidopsis function evolved after the split of mosses and seed plants. The SPA1 ortholog from rice (OsSPA1) rescued the spa mutant phenotype in dark-grown seedlings, but did not complement any spa mutant phenotype in light-grown seedlings or in adult plants. Conclusion: Our results show that COP1 protein sequences from Physcomitrella, rice and Arabidopsis have been functionally conserved during evolution, while the SPA proteins showed considerable functional divergence. This may -a t least in part -r eflect the fact thatCOP1 is a single copy gene in seed plants, while SPA proteins are encoded by a small gene family of two to four members with possibly sub- or neofunctionalized tasks.

The VP1 transcription factor functions as both a repressor and an activator of gene expression in the developing aleurone. Vp1 activation of the anthocyanin pathway exhibits strict cell autonomy in aleurone. In contrast, Vp1-mediated repression of hydrolase genes in aleurone cells during seed development is determined by a combination of cell autonomous and cell non-autonomous signals. To analyze signaling between the embryo and aleurone during seed development, a T-B3La chromosome translocation was used to create seed that has non-concordant embryo and endosperm genotypes. We show that de-repression of an Amy-GUS reporter gene in developing vp1 mutant aleurone cells strongly depends on the presence of a viviparous embryo. Genetic ablation of the developing embryo in vp1 mutant and Vp1 seeds through the introduction of an early embryo mutation caused a similar enhancement of Amy-GUS expression in the aleurone, suggesting that the quiescent embryo present in normal seed is a critical source of inhibitory signals. Analysis of an ABA deficient vp1 vp5 double mutant indicates that ABA synthesized in the embyro interacts additively with Vp1 to prevent precocious induction of alpha-amylase genes in the aleurone of the developing seed. A lack of ABA synthesis, however, does not account for the strongly synergistic interaction between a viviparous vp1 embryo and mutant aleurone suggesting that a quiescent embyro is a source of other inhibitory signals.

The five members of the phytochrome photoreceptor family of Arabidopsis thaliana control morphogenesis differentially in response to light. Genetic analysis has identified a signaling pathway that is specifically activated by phytochrome A. A component in this pathway, SPA1 (for “suppressor of phyA-105”), functions in repression of photomorphogenesis and is required for normal photosensory specificity of phytochrome A. Molecular cloning of the SPA1 gene indicates that SPA1 is a WD (tryptophan–aspartic acid)-repeat protein that also shares sequence similarity with protein kinases. SPA1 can localize to the nucleus, suggesting a possible function in phytochrome A–specific regulation of gene expression.

Summary Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.

The Viviparous-1 (VP1) transcriptional activator of maize is required for abscisic acid induction of maturation-specific genes late in seed development leading to acquisition of desiccation tolerance and arrest in embryo growth. Here, we show that VP1 also inhibits induction of the germination-specific alpha-amylase genes in aleurone cells of the developing seed and thereby appears to be involved in preventing precocious hydrolyzation of storage compounds accumulating in the endosperm. In developing seeds of the somatically instable vp1-m2 mutant, hydrolase activity was derepressed specifically in endosperm sectors underlying vp1 mutant aleurone. A barley alpha-amylase promoter-GUS reporter construct (Amy-GUS) was induced in developing vp1 mutant aleurone cells but not in wild-type aleurone cells. Moreover, transient expression of recombinant VP1 and vp1 mutant aleurone cells strongly inhibited expression of Amy-GUS and thus effectively complemented this aspect of the mutant phenotype. VP1 specifically repressed induction of Amy-GUS by the hormone gibberellic acid in aleurone of germinating barley seeds. Deletion of the acidic transcriptional activation domain of VP1 did not affect the inhibitory activity, indicating that VP1 has a discrete repressor function. Hence, physically combining activator and repressor functions in one protein may provide a mechanism to integrate the control of two normally consecutive developmental programs, seed maturation and seed germination.