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Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein–DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female–male determination and female–bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

In most grapevine planting regions, especially in south of China, plenty of rainfall and high water level underground are the characteristic of the area, a series of problem during fruit ripening easily caused poor color quality. Thereby affecting fruit quality, yield and economic benefits. The accumulation of anthocyanin is regulated by transcriptional regulatory factor and a series of cultivation measures, root restriction can make plants in the environment of stress and stress relief, root restriction induced the higher expression of VvMYB15 and VvWRKY40, and consistent with anthocyanin accumulation. Whether and how root restriction-inducible VvMYB15 and VvWRKY40 transcription factor regulate anthocyanin synthesis in grape berry is still unclear. In this study, we identified that the transient overexpression of VvMYB15 and VvWRKY40 alone or both in strawberry fruits and grape berries can promote anthocyanin accumulation and increase the expression level of anthocyanin biosynthetic genes, indicating VvMYB15 and VvWRKY40 play a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both VvMYB15 and VvWRKY40 specifically bind to the promoter region of VvF3′5′H and VvUFGT, and the expression of VvF3′5′H and VvUFGT is further activated through the heterodimer formation between VvMYB15 and VvWRKY40. Finally, we confirmed that VvMYB15 promoted anthocyanin accumulation by interacting with VvWRKY40 in grape berries, our findings provide insights into a mechanism involving the synergistic regulation of root restriction-dependent coloration and biosynthesis via a VvMYB15 and VvWRKY40 alone or both in grape berries.

Anthocyanin accumulation is transcriptionally regulated by the MYB–bHLH–WD40 complex. Light is indispensable for anthocyanin accumulation, and light-inducible MYB and HY5 were considered to promote anthocyanin accumulation in many fruits. Whether and how light-inducible bHLH transcription factor and HY5 regulate anthocyanin synthesis in strawberry is unknown. In this study, we identified a bHLH transcription factor, FvbHLH9, which was induced by light as well as FvHY5, and found that, similar to FvHY5, the transient overexpression and interference FvbHLH9 in strawberry fruits can promote and decrease anthocyanin accumulation, respectively, indicating FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both FvHY5 and FvbHLH9 specifically bind to the promoter region of some key enzyme genes, including FvDFR, and the expression of FvDFR was activated through the heterodimer formation between FvHY5 and FvbHLH9. Finally, we confirmed that FvbHLH9-promoted anthocyanin accumulation is dependent on HY5–bHLH heterodimerisation in Arabidopsis. Our findings provide insights into a mechanism involving the synergistic regulation of light-dependent coloration and anthocyanin biosynthesis via a HY5–bHLH heterodimer formed by the interaction of FvHY5 and FvbHLH9 in strawberry fruits.

Flavonoids belong to the family of polyphenolic secondary metabolites and contribute to fruit quality traits. It has been shown that MBW complexes (MYB-bHLH-WD40) regulate the flavonoids biosynthesis in different plants, but only a limited number of MBW complexes have been identified in strawberry species in general. In this study, we identified 112 R2R3-MYB proteins in woodland strawberry; 12 of them were found to have potential functions in regulating flavonoids biosynthesis by phylogenetic analysis. qRT-PCR assays showed that FvMYB3, FvMYB9, FvMYB11, FvMYB22, FvMYB64, and FvMYB105 mostly expressed at green stage of fruit development, aligned with proanthocyanidins accumulation; FvMYB10 and FvMYB41 showed higher expression levels at turning and ripe stages, aligned with anthocyanins accumulation. These results suggest that different MYBs might be involved in flavonoids biosynthesis at specific stages. Furthermore, FvMYB proteins were demonstrated to interact with FvbHLH proteins and induce expression from the promoters of CHS2 and DFR2 genes, which encode key enzymes in flavonoids biosynthesis. The co-expression of FvMYB and FvbHLH proteins in strawberry fruits also promoted the accumulation of proanthocyanidins. These findings confirmed and provided insights into the biofunction of MBW components in the regulation of flavonoid biosynthesis in woodland strawberry.

Light is a key environmental cue that fundamentally regulates plant growth and development, which is mediated by the multiple photoreceptors including the blue light (BL) photoreceptor cryptochrome 1 (CRY1). The signaling mechanism of Arabidopsis thaliana CRY1 involves direct interactions with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 1 and stabilization of COP1 substrate ELONGATED HYPOCOTYL 5 (HY5). H2A.Z is an evolutionarily conserved histone variant, which plays a critical role in transcriptional regulation through its deposition in chromatin catalyzed by SWR1 complex. Here we show that CRY1 physically interacts with SWC6 and ARP6, the SWR1 complex core subunits that are essential for mediating H2A.Z deposition, in a BL-dependent manner, and that BL-activated CRY1 enhances the interaction of SWC6 with ARP6. Moreover, HY5 physically interacts with SWC6 and ARP6 to direct the recruitment of SWR1 complex to HY5 target loci. Based on previous studies and our findings, we propose that CRY1 promotes H2A.Z deposition to regulate HY5 target gene expression and photomorphogenesis in BL through the enhancement of both SWR1 complex activity and HY5 recruitment of SWR1 complex to HY5 target loci, which is likely mediated by interactions of CRY1 with SWC6 and ARP6, and CRY1 stabilization of HY5, respectively.

Cryptochromes (CRYs) are blue light photoreceptors that mediate a variety of light responses in plants and animals, including photomorphogenesis, flowering, and circadian rhythms. The signaling mechanism by which Arabidopsis thaliana cryptochromes CRY1 and CRY2 promote photomorphogenesis involves direct interactions with COP1, a RING motif-containing E3 ubiquitin ligase, and its enhancer SPA1. Brassinosteroid (BR) is a key phytohormone involved in the repression of photomorphogenesis, and here, we show that the signaling mechanism of Arabidopsis CRY1 involves the inhibition of BR signaling. CRY1 and CRY2 physically interact with BES1-INTERACTING MYC-LIKE1 (BIM1), a basic helix-loop-helix protein. BIM1, in turn, interacts with and enhances the activity of BRI1-EMS SUPPRESSOR1 (BES1), a master transcription factor in the BR signaling pathway. In addition, CRY1 and CRY2 interact specifically with dephosphorylated BES1, the physiologically active form of BES1 that is activated by BR in a blue light-dependent manner. The CRY1-BES1 interaction leads to both the inhibition of BES1 DNA binding activity and the repression of its target gene expression. Our study suggests that the blue light-dependent, BR-induced interaction of CRY1 with BES1 is a tightly regulated mechanism by which plants optimize photomorphogenesis according to the availability of external light and internal BR signals.

The woodland strawberry, Fragaria vesca, is a model plant for the diverse Rosaceae family, which contains many valuable fruit and ornamental crops. Light is an important external environment factor which influences all aspects of plant growth and development, including fruit ripening. Transcription regulation of light will provide insights into effect of light for fruit ripening. We treat strawberry fruit with dark or light, and by the RNA-Seq method, compare transcriptional level between dark-treated and light-treated strawberry fruits. Additionally, we detect anthocyanin and sugar accumulation in two-treated conditions of fruits. Moreover, we detect the protein change of FvMYB10, a key regulator of anthocyanin biosynthesis, under dark and light. Light can promote anthocyanin and soluble sugar accumulation in mature fruit, and also increases the expression of aroma-related genes and stabilizes FvMYB10 protein. Our findings reveal that light is essential for anthocyanin and sugar accumulation and regulates FvMYB10 at transcriptional level and post-translation level.

CRY1 interacts with HBI1 in a blue light-dependent manner. CRY1 and HBI1 antagonistically regulate expression of genes promoting cell elongation and CRY1 represses HBI1’s transcriptional activity by inhibiting its DNA binding ability., Cryptochromes (CRYs) are blue light photoreceptors that mediate various light responses in plants and animals. In Arabidopsis, there are two homologous CRYs, CRY1 and CRY2, which mediate blue light inhibition of hypocotyl elongation. It is known that CRY2 interacts with CIB1, a basic helix–loop–helix (bHLH) transcriptional factor, to regulate transcription and floral induction. In this study, we performed yeast two-hybrid screening and identified CIB1 as a CRY1-interacting protein. Moreover, we demonstrated that CRY1 physically interacted with the close homolog of CIB1, HBI1, which is known to act downstream of brassinosteroid (BR) and gibberellin acid (GA) signaling pathways to promote hypocotyl elongation, in a blue light-dependent manner. Transgenic and genetic interaction studies showed that overexpression of HBI1 resulted in enhanced hypocotyl elongation under blue light and that HBI1 acted downstream of CRYs to promote hypocotyl elongation. Genome-wide gene expression analysis indicated that CRYs and HBI1 antagonistically regulated the expression of genes involved in regulating cell elongation. Moreover, we demonstrated that CRY1–HBI1 interaction led to inhibition of HBI1’s DNA binding activity and its target gene expression. Together, our results suggest that HBI1 acts as a new CRY1-interacting protein and that the signaling mechanism of CRY1 involves repression of HBI1 transcriptional activity by direct CRY1–HBI1 interaction.

Arabidopsis CRY1 and phyB are the primary blue and red light photoreceptors mediating blue and red light inhibition of hypocotyl elongation, respectively. Auxin is a pivotal phytohormone involved in promoting hypocotyl elongation. CRY1 and phyB interact with and stabilize auxin/indole acetic acid proteins (Aux/IAAs) to inhibit auxin signaling. The present study investigated whether photoreceptors might interact directly with Auxin Response Factors (ARFs) to regulate auxin signaling. Protein-protein interaction studies demonstrated that CRY1 and phyB interact physically with ARF6 and ARF8 through their N-terminal domains in a blue and red light-dependent manner, respectively. Moreover, the N-terminal DNA-binding domain of ARF6 and ARF8 is involved in mediating their interactions with CRY1. Genetic studies showed that ARF6 and ARF8 act partially downstream from CRY1 and PHYB to regulate hypocotyl elongation under blue and red light, respectively. Chromatin immunoprecipitation-PCR assays demonstrated that CRY1 and phyB mediate blue and red light repression of the DNA-binding activity of ARF6 and ARF6-target gene expression, respectively. Altogether, the results herein suggest that the direct repression of auxin-responsive gene expression mediated by the interactions of CRY1 and phyB with ARFs constitutes a new layer of the regulatory mechanisms by which light inhibits auxin-induced hypocotyl elongation.

Eggplant is rich in anthocyanins, which are the major secondary metabolites and beneficial to human health. We discovered that the anthocyanin biosynthesis of eggplant cultivar 'Lanshan Hexian' was regulated by light. In this study, we isolated two blue light receptor genes, SmCRY1 and SmCRY2, and negative/positive anthocyanin regulatory factors SmCOP1 and SmHY5 from eggplant. In terms of transcript levels, SmCRY1, SmCRY2 and SmHY5 were up-regulated by light, while SmCOP1 was down-regulated. Subsequently, the four genes were functionally complemented in phenotype of corresponding mutants, indicating that they act as counterparts of Arabidopsis genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that SmCRY1 and SmCRY2 interact with SmCOP1 in a blue-light-dependent manner. It also obtained the result that SmCOP1 interacts with SmHY5 and SmMYB1. Furthermore, using yeast one-hybrid assay, we found that SmHY5 and SmMYB1 both bind the promoters of anthocyanin biosynthesis structural genes (SmCHS and SmDFR). Taken together, blue-light-triggered CRY1/CRY2-COP1 interaction creates the condition that HY5 and MYB1 combine with the downstream anthocyanin synthesis genes (CHS and DFR) in eggplant. Our finding provides a new working model by which light controls anthocyanin accumulation in eggplant.

Figure S1. Seven polymorphic markers between WT and the M DNA pool (PDF 71 kb)

Table S1. Primers used for mapping, cloning and qPCR. (PDF 186â kb) (PDF 185 kb)

Figure S2. Partial results of linkage analysis with indel-CsUp demonstrating that this marker is co-segregated with the upward-pedicel phenotype (PDF 91 kb)

In cucumber (Cucumis sativus L.), the differentiation and development of female flowers are important processes that directly affect the fruit yield and quality. Sex differentiation is mainly controlled by three ethylene synthase genes, F (CsACS1G), M (CsACS2), and A (CsACS11). Thus, ethylene plays a key role in the sex differentiation in cucumber. The "one-hormone hypothesis" posits that F and M regulate the ethylene levels and initiate female flower development in cucumber. Nonetheless, the precise molecular mechanism of this process remains elusive. To investigate the mechanism by which F and M regulate the sex phenotype, three cucumber near-isogenic lines, namely H34 (FFmmAA, hermaphroditic), G12 (FFMMAA, gynoecious), and M12 (ffMMAA, monoecious), with different F and M loci were generated. The transcriptomic analysis of the apical shoots revealed that the expression of the B-class floral homeotic genes, CsPI (Csa4G358770) and CsAP3 (Csa3G865440), was immensely suppressed in G12 (100% female flowers) but highly expressed in M12 (∼90% male flowers). In contrast, CAG2 (Csa1G467100), which is an AG-like C-class floral homeotic gene, was specifically highly expressed in G12. Thus, the initiation of female flowers is likely to be caused by the downregulation of B-class and upregulation of C-class genes by ethylene production in the floral primordium. Additionally, CsERF31, which was highly expressed in G12, showed temporal and spatial expression patterns similar to those of M and responded to the ethylene-related chemical treatments. The biochemical experiments further demonstrated that CsERF31 could directly bind the promoter of M and promote its expression. Thus, CsERF31 responded to the ethylene signal derived from F and mediated the positive feedback regulation of ethylene by activating M expression, which offers an extended "one-hormone hypothesis" of sex differentiation in cucumber.

Light and the heterotrimeric G-protein are known to antagonistically regulate photomorphogenesis in Arabidopsis. However, whether light and G-protein coordinate the regulation of photomorphogenesis is largely unknown. Here we show that the blue light photoreceptor cryptochrome 1 (CRY1) physically interacts with the G-protein β subunit, AGB1, in a blue light-dependent manner. We also show that AGB1 directly interacts with HY5, a basic leucine zipper transcriptional factor that acts as a critical positive regulator of photomorphogenesis, to inhibit its DNA-binding activity. Genetic studies suggest that CRY1 acts partially through AGB1, and AGB1 acts partially through HY5 to regulate photomorphogenesis. Moreover, we demonstrate that blue light-triggered interaction of CRY1 with AGB1 promotes the dissociation of HY5 from AGB1. Our results suggest that the CRY1 signaling mechanism involves positive regulation of the DNA-binding activity of HY5 mediated by the CRY1–AGB1 interaction, which inhibits the association of AGB1 with HY5. We propose that the antagonistic regulation of HY5 DNA-binding activity by CRY1 and AGB1 may allow plants to balance light and G-protein signaling and optimize photomorphogenesis.

Proper timing of flowering is essential for reproduction of plants. Although it is well known that both light and gibberellin (GA) signaling play critical roles in promoting flowering in Arabidopsis thaliana, whether and how they are integrated to regulate flowering remain largely unknown. Here, we show through biochemical studies that DELLA proteins physically interact with CONSTANS (CO). Furthermore, the interaction of CO with NF-YB2 is inhibited by the DELLA protein, RGA. Our findings suggest that regulation of flowering by GA signaling in leaves under long days is mediated, at least in part, through repression of DELLA proteins on CO, providing a molecular link between DELLA proteins, key components in GA signaling pathway, and CO, a critical flowering activator in photoperiod signaling pathway.

Fundamental to plant and animal development is the regulated balance between cell proliferation and differentiation, a process intimately tied to cell cycle regulation. In Arabidopsis, mutations in TSO1, whose animal homolog is LIN54, resulted in severe developmental abnormalities both in shoot and root, including shoot meristem fasciation and reduced root meristematic zone. The molecular mechanism that could explain the tso1 mutant phenotype is absent. Through a genetic screen, we identified 32 suppressors that map to the MYB3R1 gene, encoding a conserved cell cycle regulator. Further analysis indicates that TSO1 transcriptionally represses MYB3R1, and the ectopic MYB3R1 activity mediates the tso1 mutant phenotype. Since animal homologs of TSO1 and MYB3R1 are components of a cell cycle regulatory complex, the DREAM complex, we tested and showed that TSO1 and MYB3R1 coimmunoprecipitated in tobacco leaf cells. Our work reveals a conserved cell cycle regulatory module, consisting of TSO1 and MYB3R1, for proper plant development.

Light is a key environmental cue that inhibits hypocotyl cell elongation through the blue and red/far-red light photoreceptors cryptochrome- and phytochrome-mediated pathways in Arabidopsis. In contrast, as a pivotal endogenous phytohormone auxin promotes hypocotyl elongation through the auxin receptors TIR1/AFBs-mediated degradation of AUX/IAA proteins (AUX/IAAs). However, the molecular mechanisms underlying the antagonistic interaction of light and auxin signaling remain unclear. Here, we report that light inhibits auxin signaling through stabilization of AUX/IAAs by blue and red light-dependent interactions of cryptochrome 1 (CRY1) and phytochrome B with AUX/IAAs, respectively. Blue light-triggered interactions of CRY1 with AUX/IAAs inhibit the associations of TIR1 with AUX/IAAs, leading to the repression of auxin-induced degradation of these proteins. Our results indicate that photoreceptors share AUX/IAAs with auxin receptors as the same direct downstream signaling components. We propose that antagonistic regulation of AUX/IAA protein stability by photoreceptors and auxin receptors allows plants to balance light and auxin signals to optimize their growth.

Eggplant is rich in anthocyanins, which are one of the important secondary metabolites and beneficial to human health. In this study, four anthocyanin biosynthesis genes, including chalcone synthase (SmCHS), chalcone isomerase (SmCHI), flavanone 3-hydroxylase (SmF3H) and dihydroflavonol 4-reductase (SmDFR), were isolated from eggplant. Their expression profiles were investigated along with other two structural genes (SmF3′5′H and SmANS) in different tissues, bagging, and low-temperature treatments. The highest expression levels were observed in peels except for SmF3H which was detected in stems, and SmF3H was also the exclusive gene that did not show correlation with anthocyanin content. Unlike purple peels of the control, the bagged fruits displayed white peels and had no anthocyanin accumulation, because of the expression of SmCHI, SmF3′5′H, SmDFR, and SmANS totally depended on light. As low temperature stimulates anthocyanin accumulation, all the six anthocyanin biosynthesis genes were up-regulated in cold stress experiment, and SmCHS increased most obviously. Moreover, early biosynthesis genes (SmCHS, SmCHI, and SmF3H) responded earlier than late biosynthesis genes (SmF3′5′H, SmDFR, and SmANS) under low temperature. Subcellular localization suggested that the enzymes encoded by the genes cloned in this study were all located in cytosol and nucleus. To further characterize the function, their ectopic expression in Arabidopsis was performed, and overexpression lines displayed higher anthocyanin accumulation in stems and siliques. The present study provides insight into anthocyanin biosynthesis in eggplant and may facilitate genetic engineering for improvement of the anthocyanin content in plants.

Cryptochromes (CRYs) are blue-light photoreceptors that mediate various light responses in plants and animals. The signaling mechanism by which CRYs regulate light responses involves their physical interactions with COP1. Here, we report that CRY1 interacts physically with SPA1 in a blue-light-dependent manner. SPA acts genetically downstream from CRYs to regulate light-controlled development. Blue-light activation of CRY1 attenuates the association of COP1 with SPA1 in both yeast and plant cells. These results indicate that the blue-light-triggered CRY1–SPA1 interaction may negatively regulate COP1, at least in part, by promoting the dissociation of COP1 from SPA1. This interaction and consequent dissociation define a dynamic photosensory signaling mechanism.

The stomatal pores of higher plants enable gaseous exchange into and out of leaves for photosynthesis and evaporation. Stomatal opening is induced by both blue and red lights. It is shown that blue light-induced stomatal opening is mediated by the blue light receptor phototropins (PHOT1 and PHOT2) and cryptochromes (CRY1 and CRY2). However, whether phytochrome B (phyB) is involved in red light regulation of stomatal opening remains largely unclear. Here, we report a positive role for Arabidopsis (Arabidopsis thaliana) phyB in the regulation of red light-induced stomatal opening. The phyB mutant stomata displayed a reduced red light response, whereas stomata of the phyB-overexpressing plants displayed a hypersensitive response to red light. In addition, stomata of the cry1 cry2 phyB, phot1 phot2 phyB, and cry1 phyA phyB triple mutant plants showed more reduced light response than those of the single or double mutant plants under white light, implying that phyB acts in concert with phyA, CRY, and PHOT in light regulation of stomatal opening. Stomata of phyB cop1 mutant opened less wide than those of the cop1 mutant, and stomata of the pif3 pif4 mutant opened wider than those of the wild-type, indicating that COP1, together with the PIFs (phytochrome interacting factors), may act downstream of PHYB in regulating stomatal opening. Furthermore, quantitative RT-PCR analysis showed that the expression of MYB60 was reduced in the cry1 cry2 and phyA phyB mutants under blue and red lights, respectively, but induced in the CRY1- and phyB-overexpressing plants. These results demonstrate that phyB and CRY might regulate stomatal opening, at least in part, by regulating MYB60 expression.

In Arabidopsis thaliana, the cryptochrome (CRY) blue light photoreceptors and the phytochrome (phy) red/far-red light photoreceptors mediate a variety of light responses. COP1, a RING motif–containing E3 ubiquitin ligase, acts as a key repressor of photomorphogenesis. Production of stomata, which mediate gas and water vapor exchange between plants and their environment, is regulated by light and involves phyB and COP1. Here, we show that, in the loss-of-function mutants of CRY and phyB, stomatal development is inhibited under blue and red light, respectively. In the loss-of-function mutant of phyA, stomata are barely developed under far-red light. Strikingly, in the loss-of-function mutant of either COP1 or YDA, a mitogen-activated protein kinase kinase kinase, mature stomata are developed constitutively and produced in clusters in both light and darkness. CRY, phyA, and phyB act additively to promote stomatal development. COP1 acts genetically downstream of CRY, phyA, and phyB and in parallel with the leucine-rich repeat receptor-like protein TOO MANY MOUTHS but upstream of YDA and the three basic helix-loop-helix proteins SPEECHLESS, MUTE, and FAMA, respectively. These findings suggest that light-controlled stomatal development is likely mediated through a crosstalk between the cryptochrome-phytochrome-COP1 signaling system and the mitogen-activated protein kinase signaling pathway.

Cryptochromes (CRY) are blue-light photoreceptors that mediate various light responses in plants and animals. It has long been demonstrated that Arabidopsis CRY (CRY1 and CRY2) C termini (CCT1 and CCT2) mediate light signaling through direct interaction with COP1. Most recently, CRY1 N terminus (CNT1) has been found to be involved in CRY1 signaling independent of CCT1, and implicated in the inhibition of gibberellin acids (GA)/brassinosteroids (BR)/auxin-responsive gene expression. Here, we performed RNA-Seq assay using transgenic plants expressing CCT1 fused to β-glucuronidase (GUS-CCT1, abbreviated as CCT1), which exhibit a constitutively photomorphogenic phenotype, and compared the results with those obtained previously from cry1cry2 mutant and the transgenic plants expressing CNT1 fused to nuclear localization signal sequence (NLS)-tagged YFP (CNT1-NLS-YFP, abbreviated as CNT1), which display enhanced responsiveness to blue light. We found that 2,903 (67.85％) of the CRY-regulated genes are regulated by CCT1 and that 1,095 of these CCT1-regulated genes are also regulated by CNT1. After annotating the gene functions, we found that CCT1 is involved in mediating CRY1 regulation of phytohormone-responsive genes, like CNT1, and that about half of the up-regulated genes by GA/BR/auxin are down-regulated by CCT1 and CNT1, consistent with the antagonistic role for CRY1 and these phytohormones in regulating hypocotyl elongation. Physiological studies showed that both CCT1 and CNT1 are likely involved in mediating CRY1 reduction of seedlings sensitivity to GA under blue light. Furthermore, protein expression studies demonstrate that the inhibition of GA promotion of HY5 degradation by CRY1 is likely mediated by CCT1, but not by CNT1. These results give genome-wide transcriptome information concerning the signaling mechanism of CRY1, unraveling possible involvement of its C and N termini in its regulation of response of GA and likely other phytohormones.

Light, as both energy source and informational signal, profoundly influences plant growth and development during the whole life span from seed germination to flowering. Plant senses light through multiple photoreceptors including the blue/UV-A photoreceptors cryptochromes (CRY, CRY1/2) and phototropins (PHOT, PHOT1/2), the red/far-red photoreceptors phytochromes (PHY, phyA-E), and the UV-B photoreceptor UVR8. In Arabidopsis, both CRY and PHY are involved in mediating light regulation of hypocotyl elongation, cotyledon/leaf expansion, anthocyanin accumulation, flowering, stomatal development, and light entrainment of the circadian clock.

Arabidopsis phytochromes (phyA-phyE) are photoreceptors dedicated to sensing red/far-red light. Phytochromes promote photomorphogenic developments upon light irradiation via a signaling pathway that involves rapid degradation of PIFs (PHYTOCHROME INTERACTING FACTORS) and suppression of COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) nuclear accumulation, through physical interactions with PIFs and COP1, respectively. Both phyA and phyB, the two best characterized phytochromes, regulate plant photomorphogenesis predominantly under far-red light and red light, respectively. It has been demonstrated that SPA1 (SUPPRESSOR OF PHYTOCHROME A 1) associates with COP1 to promote COP1 activity and suppress photomorphogenesis. Here, we report that the mechanism underlying phyB-promoted photomorphogenesis in red light involves direct physical and functional interactions between red-light-activated phyB and SPA1. We found that SPA1 acts genetically downstream of PHYB to repress photomorphogenesis in red light. Protein interaction studies in both yeast and Arabidopsis demonstrated that the photoactivated phyB represses the association of SPA1 with COP1, which is mediated, at least in part, through red-light-dependent interaction of phyB with SPA1. Moreover, we show that phyA physically interacts with SPA1 in a Pfr-form-dependent manner, and that SPA1 acts downstream of PHYA to regulate photomorphogenesis in far-red light. This study provides a genetic and biochemical model of how photoactivated phyB represses the activity of COP1-SPA1 complex through direct interaction with SPA1 to promote photomorphogenesis in red light.

Anthocyanins are critical for plants. It is shown that the expression of genes encoding the key enzymes such as dihydroflavonol 4-reductase (DFR), UDP-Glc: flavonoid 3-O-glucosyltransferase (UF3GT), and leucoanthocyanidin dioxygenase (LDOX) in anthocyanin biosynthesis pathway is regulated by MYB75, a R2R3 MYB transcription factor. The production of anthocyanin is known to be promoted by jasmonic acid (JA) in light but not in darkness. The photoreceptors cryptochrome 1 (CRY1), phytochrome B (phyB), and phytochrome A (phyA) are also shown to mediate light promotion of anthocyanin accumulation, respectively, whereas their downstream factor COP1, a master negative regulator of photomorphogensis, represses anthocyanin accumulation. However, whether JA coordinates with photoreceptors in the regulation of anthocyanin accumulation is unknown. Here, we show that under far-red light, JA promotes anthocyanin accumulation in a phyA signaling pathway-dependent manner. The phyA mutant is hyposensitive to jasmonic acid analog methyl jasmonic acid (MeJA) under far-red light. The dominant mutant of MYB75, pap1-D, accumulates significantly higher levels of anthocyanin than wild type under far-red light, whereas knockdown of MYBs (MYB75, MYB90, MYB113, and MYB114) through RNAi significantly reduces MeJA promotion of anthocyanin accumulation. The phyA pap1-D double mutant shows reduced responsiveness to MeJA, similar to phyA mutant under far-red light. In darkness, a mutant allele of cop1, cop1-4, shows enhanced responsiveness to MeJA, but pap1-D mutant is barely responsive to MeJA. Upon MeJA application, the cop1-4 pap1-D double mutant accumulates considerably higher levels of anthocyanin than cop1-4 in darkness. Protein studies indicate that MYB75 protein is stabilized by white light and far-red light. Further gene expression studies suggest that MeJA promotes the expression of DFR, UF3GT, and LDOX genes in a phyA- and MYB75-dependent manner under far-red light. Our findings suggest that JA promotion of anthocyanin accumulation under far-red light is dependent on phyA signaling pathway, consisting of phyA, COP1, and MYB75.

In Arabidopsis thaliana, the cryptochrome and phytochrome photoreceptors act together to promote photomorphogenic development. The cryptochrome and phytochrome signaling mechanisms interact directly with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a RING motif–containing E3 ligase that acts to negatively regulate photomorphogenesis. COP1 interacts with and ubiquitinates the transcription factors that promote photomorphogenesis, such as ELONGATED HYPOCOTYL5 and LONG HYPOCOTYL IN FAR-RED1 (HFR1), to inhibit photomorphogenic development. Here, we show that COP1 physically interacts with PIF3-LIKE1 (PIL1) and promotes PIL1 degradation via the 26S proteasome. We further demonstrate that phyB physically interacts with PIL1 and enhances PIL1 protein accumulation upon red light irradiation, probably through suppressing the COP1–PIL1 association. Biochemical and genetic studies indicate that PIL1 and HFR1 form heterodimers and promote photomorphogenesis cooperatively. Moreover, we demonstrate that PIL1 interacts with PIF1, 3, 4, and 5, resulting in the inhibition of the transcription of PIF direct-target genes. Our results reveal that PIL1 stability is regulated by phyB and COP1, likely through physical interactions, and that PIL1 coordinates with HFR1 to inhibit the transcriptional activity of PIFs, suggesting that PIL1, HFR1, and PIFs constitute a subset of antagonistic basic helix-loop-helix factors acting downstream of phyB and COP1 to regulate photomorphogenic development.