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.

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.

Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd.

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

The epidermis coordinates thermoresponsive growth through the phyB-PIF4-auxin pathway.

In plants, an elevation in ambient temperature induces adaptive morphological changes including elongated hypocotyls, which is predominantly regulated by a bHLH transcription factor, PIF4. Although PIF4 is expressed in all aerial tissues including the epidermis, mesophyll, and vascular bundle, its tissue-specific functions in thermomorphogenesis are not known. Here, we show that epidermis-specific expression of PIF4 induces constitutive long hypocotyls, while vasculature-specific expression of PIF4 has no effect on hypocotyl growth. RNA-Seq and qRT-PCR analyses reveal that auxin-responsive genes and growth-related genes are highly activated by epidermal, but not by vascular, PIF4. Additionally, inactivation of epidermal PIF4 or auxin signaling, and overexpression of epidermal phyB suppresses thermoresponsive growth, indicating that epidermal PIF4-auxin pathways are essential for the temperature responses. Further, we show that high temperatures increase both epidermal PIF4 transcription and the epidermal PIF4 DNA-binding ability. Taken together, our study demonstrates that the epidermis regulates thermoresponsive growth through the phyB-PIF4-auxin pathway.

High Ambient Temperature Accelerates Leaf Senescence via PHYTOCHROME-INTERACTING FACTOR 4 and 5 in Arabidopsis.

Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20°C but is inactivated more rapidly at 28°C. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.

Phytochrome and Ethylene Signaling Integration in Arabidopsis Occurs via the Transcriptional Regulation of Genes Co-targeted by PIFs and EIN3.

Plant seedlings germinating under the soil are challenged by rough soil grains that can induce physical damage and sudden exposure to light, which can induce photobleaching. Seedlings overcome these challenges by developing apical hooks and by suppressing chlorophyll precursor biosynthesis. These adaptive responses are, respectively, regulated by the phytochrome and ethylene signaling pathways via the PHYTOCHROME-INTERACTING FACTORs (PIFs) and the ETHYLENE INSENSITIVE 3 (EIN3)/EIN3-LIKE transcription factors. Although many processes downstream of phytochrome and ethylene signaling are similar, it remains unclear if and where these pathways converge. Here, we show PIFs and EIN3 induce similar changes in the transcriptome without robustly regulating each other's signaling pathways. PIFs and EIN3 target highly overlapped gene promoters and activate subsets of the co-target genes either interdependently or additively to induce plant responses. For chlorophyll biosynthesis, PIFs and EIN3 target and interdependently activate the expression of HOOKLESS1. HOOKLESS1, in turn, represses chlorophyll synthesis genes to prevent photobleaching. Thus, our results indicate an integration of the phytochrome and ethylene signaling pathways at the level of transcriptional gene regulation by two core groups of transcription factors, PIFs and EIN3.

PIF1 Regulates Plastid Development by Repressing Photosynthetic Genes in the Endodermis.

Mutations in Phytochrome Interacting Factors (PIFs) induce a conversion of the endodermal amyloplasts necessary for gravity sensing to plastids with developed thylakoids accompanied by abnormal activation of photosynthetic genes in the dark. In this study, we investigated how PIFs regulate endodermal plastid development by performing comparative transcriptome analysis. We show that both endodermal expression of PIF1 and global expression of the PIF quartet induce transcriptional changes in genes enriched for nuclear-encoded photosynthetic genes such as LHCA and LHCB. Among the 94 shared differentially expressed genes identified from the comparative transcriptome analysis, only 14 genes are demonstrated to be direct targets of PIF1, and most photosynthetic genes are not. Using a co-expression analysis, we identified a direct target of PIF, whose expression pattern shows a strong negative correlation with many photosynthetic genes. We have named this gene REPRESSOR OF PHOTOSYNTHETIC GENES1 (RPGE1). Endodermal expression of RPGE1 rescued the elevated expression of photosynthetic genes found in the pif quadruple (pifQ) mutant and partly restored amyloplast development and hypocotyl negative gravitropism. Taken together, our results indicate that RPGE1 acts downstream of PIF1 in the endodermis to repress photosynthetic genes and regulate plastid development.

Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis.

Plants initiate senescence to shed photosynthetically inefficient leaves. Light deprivation induces leaf senescence, which involves massive transcriptional reprogramming to dismantle cellular components and remobilize nutrients. In darkness, intermittent pulses of red light can inhibit senescence, likely via phytochromes. However, the precise molecular mechanisms transducing the signals from light perception to the inhibition of senescence remain elusive. Here, we show that in Arabidopsis, dark-induced senescence requires phytochrome-interacting transcription factors PIF4 and PIF5 (PIF4/PIF5). ELF3 and phytochrome B inhibit senescence by repressing PIF4/PIF5 at the transcriptional and post-translational levels, respectively. PIF4/PIF5 act in the signalling pathways of two senescence-promoting hormones, ethylene and abscisic acid, by directly activating expression of EIN3, ABI5 and EEL. In turn, PIF4, PIF5, EIN3, ABI5 and EEL directly activate the expression of the major senescence-promoting NAC transcription factor ORESARA1, thus forming multiple, coherent feed-forward loops. Our results reveal how classical light signalling connects to senescence in Arabidopsis.

Phytochrome-interacting factors have both shared and distinct biological roles.

Phytochromes are plant photoreceptors that perceive red and far-red light. Upon the perception of light in Arabidopsis, light-activated phytochromes enter the nucleus and act on a set of interacting proteins, modulating their activities and thereby altering the expression levels of ∼10% of the organism's entire gene complement. Phytochromeinteracting factors (PIFs) belonging to Arabidopsis basic helix-loop-helix (bHLH) subgroup 15 are key interacting proteins that play negative roles in light responses. Their activities are post-translationally countered by light-activated phytochromes, which promote the degradation of PIFs and directly or indirectly inhibit their binding to DNA. The PIFs share a high degree of similarity, but examinations of pif single and multiple mutants have indicated that they have shared and distinct functions in various developmental and physiological processes. These are believed to stem from differences in both intrinsic protein properties and their gene expression patterns. In an effort to clarify the basis of these shared and distinct functions, we compared recently published genome-wide ChIP data, developmental gene expression maps, and responses to various stimuli for the various PIFs. Based on our observations, we propose that the biological roles of PIFs stem from their shared and distinct DNA binding targets and specific gene expression patterns.