The bHLH transcription factor CsPIF4 positively regulates high temperature-induced hypocotyl elongation in cucumber

High temperature-induced hypocotyl elongation is a typical thermomorphogenesis trait that may significantly affect early seedling growth and subsequent crop yield.The ambient temperature and endogenous auxin are two critical factors that regulate hypocotyl growth.However,the mechanism of temperature and auxin integration in horticultural plants remains poorly understood.In this study,the roles of the basic helix-loop-helix transcription factor CsPIF4 in regulating auxin biosynthesis genes and the auxin content in the hypocotyl of cucumber(Cucumis sativus L.)seedlings under high temperature were investigated.qRT-PCR and in situ hybridization analysis revealed that expression of CsPIF4 was enhanced in the epidermis and vascular bundles in the hypocotyl of cucumber seedlings in response to high temperature.qRT-PCR and HPLC analysis showed that CsPIF4 positively regulated transcription of the auxin biosynthesis gene CsYUC8 and the auxin content in the hypocotyl under high temperature(35℃).The CRISPR/Cas9-mediated knockout of CsPIF4 resulted in a shorter hypocotyl compared with that of the wild type,together with decreased expression of CsYUC8 and lower auxin content in response to high temperature.Furthermore,biochemical assays showed that CsPIF4 could bind directly to the G-box motif of the CsYUC8 promoter and thereby activate CsYUC8 expression.These findings provide insight into the molecular mechanism of high temperature-mediated hypocotyl elongation in cucumber.

The PRR-EC complex and SWR1 chromatin remodeling complex function cooperatively to repress nighttime hypocotyl elongation by modulating PIF4 expression in Arabidopsis

The circadian clock entrained by environmental light-dark cycles enables plants to fine-tune diurnal growth and developmental responses.Here,we show that physical interactions among evening clock components,including PSEUDO-RESPONSE REGULATOR 5(PRR5),TIMING OF CAB EXPRESSION 1(TOC1),and the Evening Complex(EC)component EARLY FLOWERING 3(ELF3),define a diurnal repressive chromatin structure specifically at the PHYTOCHROME-INTERACTING FACTOR 4(PIF4)locus in Arabidopsis.These three clock components act interdependently as well as independently to repress nighttime hypocotyl elongation,as hypocotyl elongation rate dramatically increased specifically at nighttime in the prr5-1 toc1-21 elf3-1 mutant,concomitantly with a substantial increase in PIF4 expression.Transcriptional repression of PIF4 by ELF3,PRR5,and TOC1 is mediated by the SWI2/SNF2-RELATED(SWR1)chromatin remodeling complex,which incorporates histone H2A.Z at thePIF4 locus,facilitating robust epigenetic suppression ofPIF4 during the evening.Overall,these findings demonstrate that the PRR-EC-SWR1 complex represses hypocotyl elongation at night through a distinctive chromatin domain covering PIF4 chromatin.

The DELLA-ABI4-HY5 module integrates light and gibberellin signals to regulate hypocotyl elongation

Plant growth is coordinately controlled by various environmental and hormonal signals,of which light and gibberellin(GA)signals are two critical factors with opposite effects on hypocotyl elongation.Although interactions between the light and GA signaling pathways have been studied extensively,the detailed regulatory mechanism of their direct crosstalk in hypocotyl elongation remains to be fully clarified.Previously,we reported that ABA INSENSITIVE 4(ABI4)controls hypocotyl elongation through its regulation of cellelongation-related genes,but whether it is also involved in GA signaling to promote hypocotyl elongation is unknown.In this study,we showthat promotion of hypocotyl elongation by GA is dependent on ABI4 activation.DELLAs interact directly with ABI4 and inhibit its DNA-binding activity.In turn,ABI4 combined with ELONGATED HYPOCOTYL 5(HY5),a key positive factor in light signaling,feedback regulates the expression of the GA2ox GA catabolism genes and thus modulates GA levels.Taken together,our results suggest that the DELLA-ABI4-HY5 module may serve as a molecular link that integrates GA and light signals to control hypocotyl elongation.

Strigolactone-Regulated Hypocotyl Elongation Is Dependent on Cryptochrome and Phytochrome Signaling Pathways in Arabidopsis

Seedling development including hypocotyl elongation is a critical phase in the plant life cycle. Light regula- tion of hypocotyl elongation is primarily mediated through the blue light photoreceptor cryptochrome and red/far-red light photoreceptor phytochrome signaling pathways, comprising regulators including COP1, HY5, and phytochrome- interacting factors （PIFs）. The novel phytohormones, strigolactones, also participate in regulating hypocotyl growth. However, how strigolactone coordinates with light and photoreceptors in the regulation of hypocotyl elongation is largely unclear. Here, we demonstrate that strigolactone inhibition of hypocotyl elongation is dependent on cryp- tochrome and phytochrome signaling pathways. The photoreceptor mutants cry1 cry2, phyA, and phyB are hyposensi- tive to strigolactone analog GR24 under the respective monochromatic light conditions, while cop1 and pifl pif3 pif4 pif5 （pifq） quadruple mutants are hypersensitive to GR24 in darkness. Genetic studies indicate that the enhanced respon- siveness of cop1 to GR24 is dependent on HY5 and MAX2, while that of pifq is independent of HY5. Further studies demonstrate that GR24 constitutively up-regulates HY5 expression in the dark and light, whereas GR24-promoted HY5 protein accumulation is light- and cryptochrome and phytochrome photoreceptor-dependent. These results suggest that the light dependency of strigolactone regulation of hypocotyl elongation is likely mediated through MAX2-dependent promotion of HY5 expression, light-dependent accumulation of HY5, and PIF-regulated components.

Arabidopsis NF-YCs Mediate the Light-Controlled Hypocotyl Elongation via Modulating Histone Acetylation

Light is a crucial environmental signal that promotes photomorphogenesis, the developmental process with a series of light-dependent alterations for plants to adapt various external challenges. Chromatin modification has been proposed to be involved in such light-mediated growth, but the underlying mecha- nism is still elusive. In this study, we identified four Arabidopsis thaliana Nuclear Factor-YC homologs, NF- YCl, NF-YC3, NF-YC4, and NF-YC9 （NF-YCs）, which function redundantly as repressors of light-controlled hypocotyl elongation via histone deacetylation. Obvious etiolation phenotypes are observed in NF-YCs loss-of-function mutant seedlings grown under light conditions, including significant elongated hypocotyls and fewer opened cotyledons. We found that NF-YCs interact with histone deacetylase HDA15 in the light, co-target the promoters of a set of hypocotyl elongation-related genes, and modulate the levels of histone H4 acetylation on the associated chromatins, thus repressing gene expression. In contrast, NF-YC-HDA15 complex is dismissed from the target genes in the dark, resulting in increased level of H4 acetylation and consequent etiolated growth. Further analyses revealed that transcriptional repression activity of NF-YCs on the light-controlled hypocotyl elongation partially depends on the deacetylation activity of HDA15, and loss of HDA15 function could rescue the short-hypocotyl phenotype of NF-YCs overexpression plants. Taken together, our results indicate that NF-YCl, NF-YC3, NF-YC4, and NF-YC9 function as tran- scriptional co-repressors by interacting with HDA15 to inhibit hypocotyl elongation in photomorphogen- esis during the early seedling stage. Our findings highlight that NF-YCs can modulate plant development in response to environmental cues via epigenetic regulation.

The G-Protein β Subunit AGB1 Promotes Hypocotyl Elongation through Inhibiting Transcription Activation Function of BBX21 in Arabidopsis

Hypocotyl development in Arabidopsis thaliana is regulated by light and endogenous hormonal cues, mak- ing it an ideal model to study the interplay between light and endogenous growth regulators. BBX21, a B-box （BBX）-Iike zinc-finger transcription factor, integrates light and abscisic acid signals to regulate hypocotyl elongation in Arabidopsis. Heterotrimeric G-proteins are pivotal regulators of plant development. The short hypocotyl phenotype of the G-protein I^-subunit （AGB1） mutant （agbl-2） has been previously identified, but the precise role of AGB1 in hypocotyl elongation remains enigmatic. Here, we show that AGB1 directly interacts with BBX21, and the short hypocotyl phenotype of agbl-2 is partially suppressed in agb1-2bbx21-1 double mutant. BBX21 functions in the downstream of AGB1 and overexpression of BBX21 in agbl-2 causes a more pronounced reduction in hypocotyl length, indicating that AGB1 plays an oppositional role in relation to BBX21 during hypocotyl development. Furthermore, we demonstrate that the C-terminal region of BBX21 is important for both its intracellular localization and its transcriptional activation activity that is inhibited by interaction with AGB1. ChiP assays showed that BBX21 specifically associates with its own promoter and with those of BBX22, HY5, and GA2oxl. which is not altered in agbl-2. These data suggest that the AGB1-BBX21 interaction only affects the transcrip- tional activation activity of BBX21 but has no effect on its DNA binding ability. Taken together, our data demonstrate that AGB1 positively promotes hypocotyl elongation through repressing BBX21 activity.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 Protein Forms an SCF Ubiquitin Ligase and Regulates Hypocotyl Elongation

The regulation of protein turnover by the ubiquitin proteasome system （UPS） is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome （CSN）, regulates ＇cullin-ring＇ E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 （COP9 INTERACTING F-BOX KELCH 1）. We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteoly- sis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFKl-depleted seedlings, while complete loss of CSN activity sup- presses the Iong-hypocotyl phenotype of CFKl-overexpressing seedlings. We propose that CFK1 （and its regulation by CSN） is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Clathrin light chains regulate hypocotyl elongation by affecting the polarization of the auxin transporter PIN3 in Arabidopsis

PIN-FORMED(PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers.Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.

Candidate genes conferring ethylene-response in cultivated peanuts determined by BSA-seq and fine-mapping

Ethylene plays essential roles in plant growth,development and stress responses.The ethylene signaling pathway and molecular mechanism have been studied extensively in Arabidopsis and rice but limited in peanuts.Here,we established a sand-culture method to screen pingyangmycin mutagenized peanut lines based on their specific response to ethylene(“triple response”).An ethylene-insensitive mutant,inhibition of peanut hypocotyl elongation 1(iph1),was identified that showed reduced sensitivity to ethylene in both hypocotyl elongation and root growth.Through bulked segregant analysis sequencing,a major gene related to iph1,named AhIPH1,was preliminarily mapped at the chromosome Arahy.01,and further narrowed to a 450-kb genomic region through substitution mapping strategy.A total of 7014 genes were differentially expressed among the ACC treatment through RNA-seq analysis,of which only the Arahy.5BLU0Q gene in the candidate mapping interval was differentially expressed between WT and mutant iph1.Integrating sequence variations,functional annotation and transcriptome analysis revealed that a predicated gene,Arahy.5BLU0Q,encoding SNF1 protein kinase,may be the candidate gene for AhIPH1.This gene contained two single-nucleotide polymorphisms at promoter region and was more highly expressed in iph1 than WT.Our findings reveal a novel ethylene-responsive gene,which provides a theoretical foundation and new genetic resources for the mechanism of ethylene signaling in peanuts.

AFP2 Coordinates the Activity of PIF7 for Thermomorphogenesis in Arabidopsis Seedlings

Ambient temperature induces the hypocotyl elongation of seedling,called as thermomorphogenesis.It has been reported that the bHLH transcriptional factor PIF7 acts as the critical component to modulate plant thermomorphogenesis,but the underlying mechanism remains elusive.The phytohormone abscisic acid(ABA)suppresses the hypocotyl elongation under high temperature(HT)stress.As the ABI5 binding protein,AFP2 acts as the negative factor to control ABA signaling.In this study,we first identified AFP2 as the interaction protein of PIF7 in vitro and in vivo.Phenotype analysis revealed that overexpressing AFP2 reduced the hypocotyl elongation,while loss-of-function afp2 mutant showed longer hypocotyl under HT.Consistently,overexpressing AFP2 impaired the transactivation effect of PIF7 on auxin biosynthesis related genes YUC8 and IAA19,which possibly resulted into the shorter hypocotyl in the transgenic line overexpressing AFP2 or co-overexpressing AFP2 and PIF7.Thus,these data suggest that AFP2 suppressed PIF7 activity to suppress hypocotyl elongation.Furthermore,we found that HT gradually induced the degradation of AFP2 that possibly released the inhibitory effect of AFP2 on PIF7,thus induced hypocotyl elongation under HT.Taken together,our result reveals the novel function of AFP2 in coordinating thermomorphogenesis through sophistically modulating PIF7 activity.

Effect of Natural Brassinolide on Germination of Ailanthus altissima Seeds

The germination capacity of Ailanthus altissima seeds improved after the seeds were soaked with different concentrations of natural brassinolide （NBR）. The germination rate and germination energy of the seeds increased by 17.6% and 18.8%, and the mean germination speed （i.e., germination time） of the seeds was shortened by 1.4 d under the optimal concentration （0.4 mg·L^-1） treatment, compared with the control. After hypocotyls of A.altissima were treated with NBR, the elongation of the hypocotyls increased. Among different concentrations of the NBR, 0.4 mg·L^-1 NBR appeared to be the optimal concentration for the elongation of A.altissima hypocotyls.

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. H2 A.Z is an evolutionarily conserved H2 A variant which plays essential roles in transcriptional regulation. The replacement of H2 A with H2 A.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 coregulate 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 tryptophandependent 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 H2 A.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.

Light promotes jasmonate biosynthesis to regulate photomorphogenesis in Arabidopsis

Light acts as the pivotal external environment cue to modulate plant growth and development. Seeds germinate in the soil without light to undergo skotomorphogenesis with rapidly elongating hypocotyls that facilitate emergence from the soil, while seedlings upon light exposure undergo photomorphogenesis with significantly inhibited hypocotyl elongation that benefits plants to stand up firmly and cope with the changing environment. In this study, we demonstrate that light promotes jasmonate(JA)biosynthesis to inhibit hypocotyl elongation and orchestrate seedling photomorphogenesis in Arabidopsis. We showed that JAinhibition on hypocotyl elongation is dependent on JA receptor COI1 and signaling components such as repressor proteins JAZs and transcription activators MYC2/MYC3/MYC4. Furthermore, we found that MYC2/MYC3/MYC4 activate the expression of photomorphogenesis regulator HY5 to repress cell elongation-related genes(such as SAUR62 and EXP2) essential for seedling photomorphogenesis. Our findings provide a novel insight into molecular mechanisms underlying how plants integrate light signal with hormone pathway to establish seedling photomorphogenesis.

NF-YCs modulate histone variant H2A.Z deposition to regulate photomorphogenic growth in Arabidopsis

Inplants,lightsignalstriggeraphotomorphogenic program involving transcriptome changes, epigenetic regulation, and inhibited hypocotyl elongation. The evolutionarily conserved histone variant H2 A.Z, which functions in transcriptional regulation, is deposited in chromatin by the SWI2/SNF2-RELATED 1 complex(SWR1 c). However, the role of H2 A.Z in photomorphogenesis and its deposition mechanism remain unclear. Here, we show that in Arabidopsis thaliana, H2 A.Z deposition at its target loci is induced by light irradiation via NUCLEAR FACTOR-Y, subunit C(NF-YC) proteins, thereby inhibiting photomorphogenic growth. NF-YCs physically interact with ACTIN-RELATED PROTEIN6(ARP6), a key component of the SWR1 c that is essential for depositing H2 A.Z, in a lightdependent manner. NF-YCs and ARP6 function together as negative regulators of hypocotyl growth by depositing H2 A.Z at their target genes during photomorphogenesis. Our findings reveal an important role for the histone variant H2 A.Z in photomorphogenic growth and provide insights into a novel transcription regulatory node that mediates H2 A.Z deposition to control plant growth in response to changing light conditions.

SD3, an Arabidopsis thaliana Homolog of TIM21, Affects Intracellular ATP Levels and Seedling Development

It is poorly understood how plants control their growth by cell division, elongation, and differentiation. We have characterized a seedling-lethal mutant segregation distortion 3 （sd3） that showed a very dwarf phenotype when grown in the light and, in the dark, had short hypocotyls with reduced ploidy levels. The corresponding gene of SD3 encodes a protein with high similarity to yeast translocase on the inner mitochondrial membrane 21 （TIM21）, which is a component of the TIM23 complex. Indeed, SD3 protein fused to GFP localized in the mitochondria. SD3 overexpression increased cotyledon size in the light and hypocotyl thickness in the dark. The expression of genes for several subunits of the respiratory-chain complexes III and IV was up-regulated in SD3-overexpressing plants. Furthermore, these plants showed high levels of ATP whereas those of sd3 were low. These results suggested that SD3 induced an increase in cell size by raising the expression of the respiratory-chain subunit genes and hence increased the intracellular ATP levels, We propose that intracellular ATP levels regulated by mitochondria control plant organ size.