ADA2b acts to positively regulate blue light-mediated photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) act as blue light photoreceptors to regulate various plant physiological processes including photomorphogenesis and repair of DNA double strand breaks (DSBs). ADA2b is a conserved transcription co-activator that is involved in multiple plant developmental processes. It is known that ADA2b interacts with CRYs to mediate blue light-promoted DSBs repair. Whether ADA2b may participate in CRYs-mediated photomorphogenesis is unknown. Here we show that ADA2b acts to inhibit hypocotyl elongation and hypocotyl cell elongation in blue light. We found that the SWIRM domain-containing C-terminus mediates the blue light-dependent interaction of ADA2b with CRYs in blue light. Moreover, ADA2b and CRYs act to co-regulate the expression of hypocotyl elongation-related genes in blue light. Based on previous studies and these results, we propose that ADA2b plays dual functions in blue light-mediated DNA damage repair and photomorphogenesis.

Arabidopsis cryptochromes interact with SOG1 to promote the repair of DNA double-strand breaks.

Cryptochromes (CRYs) are blue light (BL) photoreceptors to regulate a variety of physiological processes including DNA double-strand break (DSB) repair. SUPPRESSOR OF GAMMA RADIATION 1 (SOG1) acts as the central transcription factor of DNA damage response (DDR) to induce the transcription of downstream genes, including DSB repair-related genes BRCA1 and RAD51. Whether CRYs regulate DSB repair by directly modulating SOG1 is unknown. Here, we demonstrate that CRYs physically interact with SOG1. Disruption of CRYs and SOG1 leads to increased sensitivity to DSBs and reduced DSB repair-related genes' expression under BL. Moreover, we found that CRY1 enhances SOG1's transcription activation of DSB repair-related gene BRCA1. These results suggest that the mechanism by which CRYs promote DSB repair involves positive regulation of SOG1's transcription of its target genes, which is likely mediated by CRYs-SOG1 interaction.

Arabidopsis cryptochrome 1 controls photomorphogenesis through regulation of H2A.Z deposition.

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.

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins regulate gibberellin signaling and photomorphogenesis in Arabidopsis.

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. In Arabidopsis (Arabidopsis thaliana), cryptochrome 1 (CRY1) mediates blue light-induced photomorphogenesis, which is characterized by reduced hypocotyl elongation and enhanced anthocyanin production, whereas gibberellin (GA) signaling mediated by the GA receptor GA-INSENSITIVE DWARF1 (GID1) and DELLA proteins promotes hypocotyl elongation and inhibits anthocyanin accumulation. Whether CRY1 control of photomorphogenesis involves regulation of GA signaling is largely unknown. Here, we show that CRY1 signaling involves the inhibition of GA signaling through repression of GA-induced degradation of DELLA proteins. CRY1 physically interacts with DELLA proteins in a blue light-dependent manner, leading to their dissociation from SLEEPY1 (SLY1) and the inhibition of their ubiquitination. Moreover, CRY1 interacts directly with GID1 in a blue light-dependent but GA-independent manner, leading to the inhibition of the interaction between GID1 with DELLA proteins. These findings suggest that CRY1 controls photomorphogenesis through inhibition of GA-induced degradation of DELLA proteins and GA signaling, which is mediated by CRY1 inhibition of the interactions of DELLA proteins with GID1 and SCFSLY1, respectively.

Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

Arabidopsis cryptochrome 1 undergoes COP1 and LRBs-dependent degradation in response to high blue light.

Arabidopsis cryptochrome 1 (CRY1) is an important blue light photoreceptor that promotes photomorphogenesis under blue light. The blue light photoreceptors CRY2 and phototropin 1, and the red/far-red light photoreceptors phytochromes B and A undergo degradation in response to blue and red light, respectively. This study investigated whether and how CRY1 might undergo degradation in response to high-intensity blue light (HBL). We demonstrated that CRY1 is ubiquitinated and degraded through the 26S proteasome pathway in response to HBL. We found that the E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1) is involved in mediating HBL-induced ubiquitination and degradation of CRY1. We also found that the E3 ubiquitin ligases LRBs physically interact with CRY1 and are also involved in mediating CRY1 ubiquitination and degradation in response to HBL. We further demonstrated that blue-light inhibitor of cryptochromes 1 interacts with CRY1 in a blue-light-dependent manner to inhibit CRY1 dimerization/oligomerization, leading to the repression of HBL-induced degradation of CRY1. Our findings indicate that the regulation of CRY1 stability in HBL is coordinated by COP1 and LRBs, which provides a mechanism by which CRY1 attenuates its own signaling and optimizes photomorphogenesis under HBL.

COP1 promotes ABA-induced stomatal closure by modulating the abundance of ABI/HAB and AHG3 phosphatases.

Plant stomata play a crucial role in leaf function, controlling water transpiration in response to environmental stresses and modulating the gas exchange necessary for photosynthesis. The phytohormone abscisic acid (ABA) promotes stomatal closure and inhibits light-induced stomatal opening. The Arabidopsis thaliana E3 ubiquitin ligase COP1 functions in ABA-mediated stomatal closure. However, the underlying molecular mechanisms are still not fully understood. Yeast two-hybrid assays were used to identify ABA signaling components that interact with COP1, and biochemical, molecular and genetic studies were carried out to elucidate the regulatory role of COP1 in ABA signaling. The cop1 mutants are hyposensitive to ABA-triggered stomatal closure under light and dark conditions. COP1 interacts with and ubiquitinates the Arabidopsis clade A type 2C phosphatases (PP2Cs) ABI/HAB group and AHG3, thus triggering their degradation. Abscisic acid enhances the COP1-mediated degradation of these PP2Cs. Mutations in ABI1 and AHG3 partly rescue the cop1 stomatal phenotype and the phosphorylation level of OST1, a crucial SnRK2-type kinase in ABA signaling. Our data indicate that COP1 is part of a novel signaling pathway promoting ABA-mediated stomatal closure by regulating the stability of a subset of the Clade A PP2Cs. These findings provide novel insights into the interplay between ABA and the light signaling component in the modulation of stomatal movement.

Photoexcited Cryptochrome2 Interacts Directly with TOE1 and TOE2 in Flowering Regulation.

Cryptochromes are photolyase-like, blue-light (BL) photoreceptors found in various organisms. Arabidopsis (Arabidopsis thaliana) cryptochromes (CRYs; CRY1, and CRY2) mediate many light responses including photoperiodic floral initiation. Cryptochromes interact with COP1 and SPA1, causing the stabilization of CONSTANS (CO) and promotion of FLOWERING LOCUS T (FT) transcription and flowering. The AP2-like transcriptional factor TOE1 negatively regulates FT expression and flowering by indirectly inhibiting CO transcriptional activation activity and directly binding to FT Here, we demonstrate that CRY1 and CRY2 physically interact with TOE1 and TOE2 in a BL-dependent manner in flowering regulation. Genetic studies showed that mutation of TOE1 and TOE2 partially suppresses the late-flowering phenotype of cry1 cry2 mutant plants. BL-triggered interactions of CRY2 with TOE1 and TOE2 promote the dissociation of TOE1 and TOE2 from CO, resulting in alleviation of their inhibition of CO transcriptional activity and enhanced transcription of FT Furthermore, we show that CRY2 represses TOE1 binding to the regulatory element within the Block E enhancer of FT These results reveal that TOE1 and TOE2 act as downstream components of CRY2, thus partially mediating CRY2 regulation of photoperiodic flowering through modulation of CO activity and FT transcription.

Blue Light Regulates Secondary Cell Wall Thickening via MYC2/MYC4 Activation of the NST1-Directed Transcriptional Network in Arabidopsis.

Secondary cell walls (SCWs) are formed in some specific types of plant cells, providing plants with mechanical strength. During plant growth and development, formation of secondary cell walls is regulated by various developmental and environmental signals. The underlying molecular mechanisms are poorly understood. In this study, we analyzed the blue light receptor cryptochrome1 (cry1) mutant of Arabidopsis thaliana for its SCW phenotypes. During inflorescence stem growth, SCW thickening in the vasculature was significantly affected by blue light. cry1 plants displayed a decline of SCW thickening in fiber cells, while CRY1 overexpression led to enhanced SCW formation. Transcriptome analysis indicated that the reduced SCW thickening was associated with repression of the NST1-directed transcription regulatory networks. Further analyses revealed that the expression of MYC2/MYC4 that is induced by blue light activates the transcriptional network underlying SCW thickening. The activation is caused by direct binding of MYC2/MYC4 to the NST1 promoter. This study demonstrates that SCW thickening in fiber cells is regulated by a blue light signal that is mediated through MYC2/MYC4 activation of NST1-directed SCW formation in Arabidopsis.

Photoexcited CRYPTOCHROME1 Interacts with Dephosphorylated BES1 to Regulate Brassinosteroid Signaling and Photomorphogenesis in Arabidopsis.

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.

Phytochrome B interacts with SWC6 and ARP6 to regulate H2A.Z deposition and photomorphogensis in Arabidopsis.

Light serves as a crucial environmental cue which modulates plant growth and development, and which is controlled by multiple photoreceptors including the primary red light photoreceptor, phytochrome B (phyB). The signaling mechanism of phyB involves direct interactions with a group of basic helix-loop-helix (bHLH) transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and the negative regulators of photomorphogenesis, COP1 and SPAs. H2A.Z is an evolutionarily conserved H2A variant which plays essential roles in transcriptional regulation. The replacement of H2A with H2A.Z is catalyzed by the SWR1 complex. Here, we show that the Pfr form of phyB physically interacts with the SWR1 complex subunits SWC6 and ARP6. phyB and ARP6 co-regulate numerous genes in the same direction, some of which are associated with auxin biosynthesis and response including YUC9, which encodes a rate-limiting enzyme in the tryptophan-dependent auxin biosynthesis pathway. Moreover, phyB and HY5/HYH act to inhibit hypocotyl elongation partially through repression of auxin biosynthesis. Based on our findings and previous studies, we propose that phyB promotes H2A.Z deposition at YUC9 to inhibit its expression through direct phyB-SWC6/ARP6 interactions, leading to repression of auxin biosynthesis, and thus inhibition of hypocotyl elongation in red light.

Arabidopsis cryptochrome 1 promotes stomatal development through repression of AGB1 inhibition of SPEECHLESS DNA-binding activity.

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. The heterotrimeric G-protein is known to regulate various physiological processes in plants and mammals. In Arabidopsis, cryptochrome 1 (CRY1) and the G-protein ß subunit AGB1 act antagonistically to regulate stomatal development. The molecular mechanism by which CRY1 and AGB1 regulate this process remains unknown. Here, we show that Arabidopsis CRY1 acts partially through AGB1, and AGB1 acts through SPEECHLESS (SPCH), a master transcription factor that drives stomatal initiation and proliferation, to regulate stomatal development. We demonstrate that AGB1 physically interacts with SPCH to block the bHLH DNA-binding domain of SPCH and inhibit its DNA-binding activity. Moreover, we demonstrate that photoexcited CRY1 represses the interaction of AGB1 with SPCH to release AGB1 inhibition of SPCH DNA-binding activity, leading to the expression of SPCH-target genes promoting stomatal development. Taken together, our results suggest that the mechanism by which CRY1 promotes stomatal development involves positive regulation of the DNA-binding activity of SPCH mediated by CRY1 inhibition of the AGB1-SPCH interaction. We propose that the antagonistic regulation of SPCH DNA-binding activity by CRY1 and AGB1 may allow plants to balance light and G-protein signaling and optimize stomatal density and pattern.

A dynamic model of UVR8 photoreceptor signalling in UV-B-acclimated Arabidopsis.

The photoreceptor UVR8 mediates numerous photomorphogenic responses of plants to UV-B wavelengths by regulating transcription. Studies with purified UVR8 and seedlings not previously exposed to UV-B have generated a model for UVR8 action in which dimeric UVR8 rapidly monomerises in response to UV-B exposure to initiate signalling. However, the mechanism of UVR8 action in UV-B-acclimated plants growing under photoperiodic conditions, where UVR8 exists in a dimer/monomer photo-equilibrium, is poorly understood. We examined UVR8 dimer/monomer status, gene expression responses, amounts of key UVR8 signalling proteins and their interactions with UVR8 in UV-B-acclimated Arabidopsis. We show that in UV-B-acclimated plants UVR8 can mediate a response to a 15-fold increase in UV-B without any increase in abundance of UVR8 monomer. Following transfer to elevated UV-B, monomers show increased interaction with both COP1, to initiate signalling and RUP2, to maintain the photo-equilibrium when the dimer/monomer cycling rate increases. Native RUP1 is present in low abundance compared with RUP2. We present a model for UVR8 action in UV-B-acclimated plants growing in photoperiodic conditions that incorporates dimer and monomer photoreception, dimer/monomer cycling, abundance of native COP1 and RUP proteins, and interactions of the monomer population with COP1, RUP2 and potentially other proteins.

Photoexcited CRY1 and phyB interact directly with ARF6 and ARF8 to regulate their DNA-binding activity and auxin-induced hypocotyl elongation in Arabidopsis.

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.

phyB Interacts with BES1 to Regulate Brassinosteroid Signaling in Arabidopsis.

Light is an important environmental factor, which mainly inhibits hypocotyl elongation through various photoreceptors. In contrast, brassinosteroids (BRs) are major hypocotyl elongation-promoting hormones in plants, which could optimize photomorphogenesis concurrent with external light. However, the precise molecular mechanisms underlying the antagonism of light and BR signaling remain largely unknown. Here we show that the Arabidopsis red light receptor phyB is involved in inhibition of BR signaling via its direct interaction with the BR transcription factor BES1. In our study, the phyB mutant displays BR hypersensitivity, which is repressed in transgenic plants overexpressing phyB, suggesting that phyB negatively regulates the BR signaling pathway. In addition, protein interaction results show that phyB directly interacts with dephosphorylated BES1, the physiologically active form of BES1 induced by BR, in a red light-dependent manner. Genetic analyses suggest that phyB may act partially through BES1 to regulate BR signaling. Transcriptomic data and quantitative real-time PCR assay further show that phyB-mediated red light inhibits BR signaling by repressing expression of BES1 target genes, including the BR biosynthesis genes DWF4, the SAUR family and the PRE family genes required for promoting cell elongation. Finally, we found that red light treatment inhibits the DNA-binding activity of BES1 and photoactivated phyB represses the transcriptional activity of BES1 under red light. Taken together, we suggest that the interaction of phyB with dephosphorylated BES1 may allow plants to balance light and BR signaling by repressing transcriptional activity of BES1 to regulate expression of its target genes.

Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.

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.

CRY1 interacts directly with HBI1 to regulate its transcriptional activity and photomorphogenesis in Arabidopsis.

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.

COP1 and phyB Physically Interact with PIL1 to Regulate Its Stability and Photomorphogenic Development in Arabidopsis.

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.

Auxin inhibits stomatal development through MONOPTEROS repression of a mobile peptide gene STOMAGEN in mesophyll.

Plants, as sessile organisms, must coordinate various physiological processes to adapt to ever-changing surrounding environments. Stomata, the epidermal pores facilitating gas and water exchange, play important roles in optimizing photosynthetic efficiency and adaptability. Stomatal development is under the control of an intrinsic program mediated by a secretory peptide gene family--namely, EPIDERMAL PATTERNING FACTOR, including positively acting STOMAGEN/EPFL9. The phytohormone brassinosteroids and environment factor light also control stomatal production. However, whether auxin regulates stomatal development and whether peptide signaling is coordinated with auxin signaling in the regulation of stomatal development remain largely unknown. Here we show that auxin negatively regulates stomatal development through MONOPTEROS (also known as ARF5) repression of the mobile peptide gene STOMAGEN in mesophyll. Through physiological, genetic, transgenic, biochemical, and molecular analyses, we demonstrate that auxin inhibits stomatal development through the nuclear receptor TIR1/AFB-mediated signaling, and that MONOPTEROS directly binds to the STOMAGEN promoter to suppress its expression in mesophyll and inhibit stomatal development. Our results provide a paradigm of cross-talk between phytohormone auxin and peptide signaling in the regulation of stomatal production.

Arabidopsis miR171-targeted scarecrow-like proteins bind to GT cis-elements and mediate gibberellin-regulated chlorophyll biosynthesis under light conditions.

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of ß-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light.