The phytochrome-interacting factor genes PIF1 and PIF4 are functionally diversified due to divergence of promoters and proteins.

Phytochrome-interacting factors (PIFs) are basic helix-loop-helix transcription factors that regulate light responses downstream of phytochromes. In Arabidopsis (Arabidopsis thaliana), eight PIFs (PIF1-8) regulate light responses, either redundantly or distinctively. Distinctive roles of PIFs may be attributed to differences in mRNA expression patterns governed by promoters or variations in molecular activities of proteins. However, elements responsible for the functional diversification of PIFs have yet to be determined. Here, we investigated the role of promoters and proteins in the functional diversification of PIF1 and PIF4 by analyzing transgenic lines expressing promoter-swapped PIF1 and PIF4, as well as chimeric PIF1 and PIF4 proteins. For seed germination, PIF1 promoter played a major role, conferring dominance to PIF1 gene with a minor contribution from PIF1 protein. Conversely, for hypocotyl elongation under red light, PIF4 protein was the major element conferring dominance to PIF4 gene with the minor contribution from PIF4 promoter. In contrast, both PIF4 promoter and PIF4 protein were required for the dominant role of PIF4 in promoting hypocotyl elongation at high ambient temperatures. Together, our results support that the functional diversification of PIF1 and PIF4 genes resulted from contributions of both promoters and proteins, with their relative importance varying depending on specific light responses.

Phytochrome B photobody components.

Phytochrome B (phyB) is a red and far-red photoreceptor that promotes light responses. Upon photoactivation, phyB enters the nucleus and forms a molecular condensate called a photobody through liquid-liquid phase separation. Phytochrome B photobody comprises phyB, the main scaffold molecule, and at least 37 client proteins. These clients belong to diverse functional categories enriched with transcription regulators, encompassing both positive and negative light signaling factors, with the functional bias toward the negative factors. The functionally diverse clients suggest that phyB photobody acts either as a trap to capture proteins, including negatively acting transcription regulators, for processes such as sequestration, modification, or degradation or as a hub where proteins are brought into close proximity for interaction in a light-dependent manner.

Shade represses photosynthetic genes by disrupting the DNA binding of GOLDEN2-LIKE1.

PHYTOCHROME-INTERACTING FACTORs (PIFs) repress photosynthetic genes partly by upregulating REPRESSOR OF PHOTOSYNTHETIC GENES 1 (RPGE1) and RPGE2. However, it is unknown how RPGEs inhibit gene expression at the molecular level. Here, we show that Arabidopsis (Arabidopsis thaliana) RPGE overexpression lines display extensive similarities to the golden2-like 1 (glk1)/glk2 double mutant at the phenotypic and transcriptomic levels, prompting us to hypothesize that there is a close molecular relationship between RPGEs and chloroplast development-regulating GLK transcription factors. Indeed, we found that RPGE1 disrupts the homodimerization of GLK1 by interacting with its dimerization domain and debilitates the DNA-binding activity of GLK1. The interaction was not restricted to the Arabidopsis RPGE1-GLK1 pair, but rather extended to RPGE-GLK homolog pairs across species, providing a molecular basis for the pale green leaves of Arabidopsis transgenic lines expressing a rice (Oryza sativa) RPGE homolog. Our discovery of RPGE-GLK regulatory pairs suggests that any condition leading to an increase in RPGE levels would decrease the expression levels of GLK target genes. Consistently, we found that shade, which upregulates the RPGE mRNA by stabilizing PIFs, represses the expression of photosynthetic genes partly by inhibiting the DNA-binding activity of GLK1. Taken together, these results indicate that RPGE-GLK regulatory pairs regulate photosynthetic gene expression downstream of PIFs.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

ABI3- and PIF1-mediated regulation of GIG1 enhances seed germination by detoxification of methylglyoxal in Arabidopsis.

Methylglyoxal (MG) is a toxic by-product of the glycolysis pathway in most living organisms and was previously shown to inhibit seed germination. MG is detoxified by glyoxalase I and II family proteins in plants. MG is abundantly produced during early embryogenesis in Arabidopsis seeds. However, the mechanism that alleviates the toxic effect of MG in maturing seeds is poorly understood. In this study, by T-DNA mutant population screening, we found that mutations in a glyoxalase I gene (named GERMINATION-IMPAIRED GLYOXALASE 1, GIG1) led to significantly impaired germination compared with wild-type seeds. Transformation of full-length GIG1 cDNA under the constitutively active cauliflower mosaic virus 35S promoter in the gig1 background completely recovered the seed germination phenotype. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that GIG1 is uniquely expressed in seeds and is upregulated by abscisic acid (ABA) and downregulated by gibberellic acid (GA) during seed germination. An ABA signaling component, ABI3, directly activated GIG1 in maturing seeds. In addition, PHYTOCHROME INTERACTING FACTOR 1 (PIF1) also plays cooperatively with ABI3 in the regulation of GIG1 expression in the early stage of imbibed seeds. Furthermore, GIG1 expression is stably silenced by epigenetic repressors such as polycomb repressor complexes. Altogether, our results indicate that light and ABA signaling cooperate to enhance seed germination by the upregulation of GIG1 to detoxify MG in maturing seeds.

Epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm.

Phytochrome B (phyB) expressed in the epidermis is sufficient to promote red light responses, including the inhibition of hypocotyl elongation and hypocotyl negative gravitropism. Nonetheless, the downstream mechanism of epidermal phyB in promoting light responses had been elusive. Here, we mutagenized the epidermis-specific phyB-expressing line (MLB) using ethyl methanesulfonate (EMS) and characterized a novel mutant allele of RRC1 (rrc1-689), which causes reduced epidermal phyB-mediated red light responses. The rrc1-689 mutation increases the alternative splicing of major clock gene transcripts, including PRR7 and TOC1, disrupting the rhythmic expression of the entire clock and clock-controlled genes. Combined with the result that MLB/prr7 exhibits the same red-hyposensitive phenotypes as MLB/rrc1-689, our data support that the circadian clock is required for the ability of epidermal phyB to promote light responses. We also found that, unlike phyB, RRC1 preferentially acts in the endodermis to maintain the circadian rhythm by suppressing the alternative splicing of core clock genes. Together, our results suggest that epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm in Arabidopsis thaliana.

PHYTOCHROME INTERACTING FACTOR8 Inhibits Phytochrome A-Mediated Far-Red Light Responses in Arabidopsis.

PHYTOCHROME INTERACTING FACTORs (PIFs) are a group of basic helix-loop-helix (bHLH) transcription factors that repress plant light responses. PIF8 is one of the less-characterized Arabidopsis (Arabidopsis thaliana) PIFs, whose putative orthologs are conserved in other plant species. PIF8 possesses a bHLH motif and an active phytochrome B motif but not an active phytochrome A motif. Consistent with this motif composition, PIF8 binds to G-box elements and interacts with the Pfr form of phyB but only very weakly, if at all, with that of phyA. PIF8 differs, however, from other PIFs in its protein accumulation pattern and functional roles in different light conditions. First, PIF8 inhibits phyA-induced seed germination, suppression of hypocotyl elongation, and randomization of hypocotyl growth orientation in far-red light, but it does not inhibit phyB-induced red light responses. Second, PIF8 protein accumulates more in far-red light than in darkness or red light. This is distinct from the pattern observed with PIF3, which accumulates more in darkness. This PIF8 accumulation pattern requires degradation of PIF8 by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in darkness, inhibition of COP1 by phyA in far-red light, and promotion of PIF8 degradation by phyB in red light. Together, our results indicate that PIF8 is a genuine PIF that represses phyA-mediated light responses.

Phytochrome B Requires PIF Degradation and Sequestration to Induce Light Responses across a Wide Range of Light Conditions.

Phytochrome B (phyB) inhibits the function of phytochrome-interacting factors (PIFs) by inducing their degradation and sequestration, but the relative physiological importance of these two phyB activities is unclear. In an analysis of published Arabidopsis thaliana phyB mutations, we identified a point mutation in the N-terminal half of phyB (phyBG111D) that abolishes its PIF sequestration activity without affecting its PIF degradation activity. We also identified a point mutation in the phyB C-terminal domain, which, when combined with a deletion of the C-terminal end (phyB990G767R), does the opposite; it blocks PIF degradation without affecting PIF sequestration. The resulting phyB proteins, phyB990G767R and phyBG111D, are equally capable of inducing light responses under continuous red light. However, phyBG111D, which exhibits only the PIF degradation activity, induces stronger light responses than phyB990G767R under white light with prolonged dark periods (i.e., diurnal cycles). In contrast, phyB990G767R, which exhibits only the PIF sequestration activity, induces stronger light responses in flickering light (a condition that mimics sunflecks). Together, our results indicate that both of these separable phyB activities are required for light responses in varying light conditions.

Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism Non-Cell-Autonomously.

Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism.

PIF1-Interacting Transcription Factors and Their Binding Sequence Elements Determine the in Vivo Targeting Sites of PIF1.

The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) binds G-box elements in vitro and inhibits light-dependent germination in Arabidopsis thaliana A previous genome-wide analysis of PIF1 targeting indicated that PIF1 binds 748 sites in imbibed seeds, only 59% of which possess G-box elements. This suggests the G-box is not the sole determinant of PIF1 targeting. The targeting of PIF1 to specific sites could be stabilized by PIF1-interacting transcription factors (PTFs) that bind other nearby sequence elements. Here, we report PIF1 targeting sites are enriched with not only G-boxes but also with other hexameric sequence elements we named G-box coupling elements (GCEs). One of these GCEs possesses an ACGT core and serves as a binding site for group A bZIP transcription factors, including ABSCISIC ACID INSENSITIVE5 (ABI5), which inhibits seed germination in abscisic acid signaling. PIF1 interacts with ABI5 and other group A bZIP transcription factors and together they target a subset of PIF1 binding sites in vivo. In vitro single-molecule fluorescence imaging confirms that ABI5 facilitates PIF1 binding to DNA fragments possessing multiple G-boxes or the GCE alone. Thus, we show in vivo PIF1 targeting to specific binding sites is determined by its interaction with PTFs and their binding to GCEs.

Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways.

Arabidopsis EARLY FLOWERING3 (ELF3) functions in modulating light input to the circadian clock, as a component of ELF3-ELF4-LUX ARRHYTHMO (LUX) evening complex. However, the role of ELF3 in stress responses remains largely unknown. In this study, we show that ELF3 enhances plants' resilience to salt stress: ELF3-overexpressing (ELF3-OX) plants are salt-tolerant, while elf3 mutants are more sensitive to salt stress. The expressions of many salt stress- and senescence-associated genes are altered in elf3-1 and ELF3-OX plants compared with wild-type. During salt stress, ELF3 suppresses factors that promote salt stress response pathways, mainly GIGANTEA (GI), at the post-translational level, and PHYTOCHROME INTERACTING FACTOR4 (PIF4), at the transcriptional level. To enhance the salt stress response, PIF4 directly downregulates the transcription of JUNGBRUNNEN1 (JUB1/ANAC042), encoding a transcription factor that upregulates the expression of stress tolerance genes, DREB2A and DELLA. Furthermore, PIF4 directly upregulates the transcription of ORESARA1 (ORE1/ANAC092) and SAG29, positive regulators of salt stress response pathways. Based on our results, we propose that ELF3 modulates key regulatory components in salt stress response pathways at the transcriptional and post-translational levels.

Functionally redundant LNG3 and LNG4 genes regulate turgor-driven polar cell elongation through activation of XTH17 and XTH24.

KEY MESSAGE: In this work, we genetically characterized the function of Arabidopsis thaliana, LONGIFOLIA (LNG1), LNG2, LNG3, LNG4, their contribution to regulate vegetative architecture in plant. We used molecular and biophysical approaches to elucidate a gene function that regulates vegetative architecture, as revealed by the leaf phenotype and later effects on flowering patterns in Arabidopsis loss-of-function mutants. As a result, LNG genes play an important role in polar cell elongation by turgor pressure controlling the activation of XTH17 and XTH24. Plant vegetative architecture is related to important traits that later influence the floral architecture involved in seed production. Leaf morphology is the primary key trait to compose plant vegetative architecture. However, molecular mechanism on leaf shape determination is not fully understood even in the model plant A. thaliana. We previously showed that LONGIFOLIA (LNG1) and LONGIFOLIA2 (LNG2) genes regulate leaf morphology by promoting longitudinal cell elongation in Arabidopsis. In this study, we further characterized two homologs of LNG1, LNG3, and LNG4, using genetic, biophysical, and molecular approaches. Single loss-of-function mutants, lng3 and lng4, do not show any phenotypic difference, but mutants of lng quadruple (lngq), and lng1/2/3 and lng1/2/4 triples, display reduced leaf length, compared to wild type. Using the paradermal analysis, we conclude that the reduced leaf size of lngq is due to decreased cell elongation in the direction of longitudinal leaf growth, and not decreased cell proliferation. This data indicate that LNG1/2/3/4 are functionally redundant, and are involved in polar cell elongation in Arabidopsis leaf. Using a biophysical approach, we show that the LNGs contribute to maintain high turgor pressure, thus regulating turgor pressure-dependent polar cell elongation. In addition, gene expression analysis showed that LNGs positively regulate the expression of the cell wall modifying enzyme encoded by a multi-gene family, xyloglucan endotransglucosylase/hydrolase (XTH). Taking all of these together, we propose that LNG related genes play an important role in polar cell elongation by changing turgor pressure and controlling the activation of XTH17 and XTH24.

Gibberellin Signaling Requires Chromatin Remodeler PICKLE to Promote Vegetative Growth and Phase Transitions.

PICKLE (PKL) is an ATP-dependent chromodomain-helicase-DNA-binding domain (CHD3) chromatin remodeling enzyme in Arabidopsis (Arabidopsis thaliana). Previous studies showed that PKL promotes embryonic-to-vegetative transition by inhibiting expression of seed-specific genes during seed germination. The pkl mutants display a low penetrance of the "pickle root" phenotype, with a thick and green primary root that retains embryonic characteristics. The penetrance of this pickle root phenotype in pkl is dramatically increased in gibberellin (GA)-deficient conditions. At adult stages, the pkl mutants are semidwarfs with delayed flowering time, which resemble reduced GA-signaling mutants. These findings suggest that PKL may play a positive role in regulating GA signaling. A recent biochemical analysis further showed that PKL and GA signaling repressors DELLAs antagonistically regulate hypocotyl cell elongation genes by direct protein-protein interaction. To elucidate further the role of PKL in GA signaling and plant development, we studied the genetic interaction between PKL and DELLAs using the hextuple mutant containing pkl and della pentuple (dP) mutations. Here, we show that PKL is required for most of GA-promoted developmental processes, including vegetative growth such as hypocotyl, leaf, and inflorescence stem elongation, and phase transitions such as juvenile-to-adult leaf and vegetative-to-reproductive phase. The removal of all DELLA functions (in the dP background) cannot rescue these phenotypes in pkl RNA-sequencing analysis using the ga1 (a GA-deficient mutant), pkl, and the ga1 pkl double mutant further shows that expression of 80% of GA-responsive genes in seedlings is PKL dependent, including genes that function in cell elongation, cell division, and phase transitions. These results indicate that the CHD3 chromatin remodeler PKL is required for regulating gene expression during most of GA-regulated developmental processes.

The Transcriptional Coregulator LEUNIG_HOMOLOG Inhibits Light-Dependent Seed Germination in Arabidopsis.

PHYTOCHROME-INTERACTING FACTOR1 (PIF1) is a basic helix-loop-helix transcription factor that inhibits light-dependent seed germination in Arabidopsis thaliana. However, it remains unclear whether PIF1 requires other factors to regulate its direct targets. Here, we demonstrate that LEUNIG_HOMOLOG (LUH), a Groucho family transcriptional corepressor, binds to PIF1 and coregulates its targets. Not only are the transcriptional profiles of the luh and pif1 mutants remarkably similar, more than 80% of the seeds of both genotypes germinate in the dark. We show by chromatin immunoprecipitation that LUH binds a subset of PIF1 targets in a partially PIF1-dependent manner. Unexpectedly, we found LUH binds and coregulates not only PIF1-activated targets but also PIF1-repressed targets. Together, our results indicate LUH functions with PIF1 as a transcriptional coregulator to inhibit seed germination.

DELLA proteins and their interacting RING Finger proteins repress gibberellin responses by binding to the promoters of a subset of gibberellin-responsive genes in Arabidopsis.

DELLA proteins, consisting of GA INSENSITIVE, REPRESSOR OF GA1-3, RGA-LIKE1 (RGL1), RGL2, and RGL3, are central repressors of gibberellin (GA) responses, but their molecular functions are not fully understood. We isolated four DELLA-interacting RING domain proteins, previously designated as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 (collectively referred to as BOIs). Single mutants of each BOI gene failed to significantly alter GA responses, but the boi quadruple mutant (boiQ) showed a higher seed germination frequency in the presence of paclobutrazol, precocious juvenile-to-adult phase transition, and early flowering, all of which are consistent with enhanced GA signaling. By contrast, BOI overexpression lines displayed phenotypes consistent with reduced GA signaling. Analysis of a gai-1 boiQ pentuple mutant further indicated that the GAI protein requires BOIs to inhibit a subset of GA responses. At the molecular level, BOIs did not significantly alter the stability of a DELLA protein. Instead, BOI and DELLA proteins are targeted to the promoters of a subset of GA-responsive genes and repress their expression. Taken together, our results indicate that the DELLA and BOI proteins inhibit GA responses by interacting with each other, binding to the same promoters of GA-responsive genes, and repressing these genes.

ABA-insensitive3, ABA-insensitive5, and DELLAs Interact to activate the expression of SOMNUS and other high-temperature-inducible genes in imbibed seeds in Arabidopsis.

Seeds monitor the environment to germinate at the proper time, but different species respond differently to environmental conditions, particularly light and temperature. In Arabidopsis thaliana, light promotes germination but high temperature suppresses germination. We previously reported that light promotes germination by repressing SOMNUS (SOM). Here, we examined whether high temperature also regulates germination through SOM and found that high temperature activates SOM expression. Consistent with this, som mutants germinated more frequently than the wild type at high temperature. The induction of SOM mRNA at high temperature required abscisic acid (ABA) and gibberellic acid biosynthesis, and ABA-insensitive3 (ABI3), ABI5, and DELLAs positively regulated SOM expression. Chromatin immunoprecipitation assays indicated that ABI3, ABI5, and DELLAs all target the SOM promoter. At the protein level, ABI3, ABI5, and DELLAs all interact with each other, suggesting that they form a complex on the SOM promoter to activate SOM expression at high temperature. We found that high-temperature-inducible genes frequently have RY motifs and ABA-responsive elements in their promoters, some of which are targeted by ABI3, ABI5, and DELLAs in vivo. Taken together, our data indicate that ABI3, ABI5, and DELLAs mediate high-temperature signaling to activate the expression of SOM and other high-temperature-inducible genes, thereby inhibiting seed germination.

Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA.

Freshly matured seeds exhibit primary dormancy, which prevents germination until environmental conditions are favorable. The establishment of dormancy occurs during seed development and involves both genetic and environmental factors that impact on the ratio of two antagonistic phytohormones: abscisic acid (ABA), which promotes dormancy, and gibberellic acid, which promotes germination. Although our understanding of dormancy breakage in mature seeds is well advanced, relatively little is known about the mechanisms involved in establishing dormancy during seed maturation. We previously showed that the SPATULA (SPT) transcription factor plays a key role in regulating seed germination. Here we investigate its role during seed development and find that, surprisingly, it has opposite roles in setting dormancy in Landsberg erecta and Columbia Arabidopsis ecotypes. We also find that SPT regulates expression of five transcription factor encoding genes: ABA-INSENSITIVE4 (ABI4) and ABI5, which mediate ABA signaling; REPRESSOR-OF-GA (RGA) and RGA-LIKE3 involved in gibberellic acid signaling; and MOTHER-OF-FT-AND-TFL1 (MFT) that we show here promotes Arabidopsis seed dormancy. Although ABI4, RGA, and MFT are repressed by SPT, ABI5 and RGL3 are induced. Furthermore, we show that RGA, MFT, and ABI5 are direct targets of SPT in vivo. We present a model in which SPT drives two antagonistic "dormancy-repressing" and "dormancy-promoting" routes that operate simultaneously in freshly matured seeds. Each of these routes has different impacts and this in turn explains the opposite effect of SPT on seed dormancy of the two ecotypes analyzed here.

A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds.

Phytochrome-interacting factor 1 (PIF1) inhibits light-dependent seed germination. The specific function of PIF1 in seed germination is partly due to its high level of expression in imbibed seeds, but the associated regulatory factors have not been identified. Here we show that mutation of the early flowering in short days (EFS) gene, encoding an H3K4 and H3K36 methyltransferase, decreases the level of H3K36me2 and H3K36me3 but not H3K4me3 at the PIF1 locus, reduces the targeting of RNA polymerase II to the PIF1 locus, and reduces mRNA expression of PIF1 in imbibed seeds. Consistently, the efs mutant geminated even under the phyBoff condition, and had an expression profile of PIF1 target genes similar to that of the pif1 mutant. Introduction of an EFS transgene into the efs mutant restored the level of H3K36me2 and H3K36me3 at the PIF1 locus, the high-level expression of PIF1 mRNA, the expression pattern of PIF1 target genes, and the light-dependent germination of these seeds. Introduction of a PIF1 transgene into the efs mutant also restored the expression pattern of PIF1 target genes and light-dependent germination in imbibed seeds, but did not restore the flowering phenotype. Taken together, our results indicate that EFS is necessary for high-level expression of PIF1 mRNA in imbibed seeds.