Transcription Factor HY5 Research Articles

CONSTANS alters the circadian clock in Arabidopsis thaliana

Plants are sessile organisms that have acquired highly plastic developmental strategies to adapt to the environment. Among these processes, the floral transition is essential to ensure reproductive success and is finely regulated by several internal and external genetic networks. The photoperiodic pathway, which controls plant response to day length, is one of the most important pathways controlling flowering. In Arabidopsis photoperiodic flowering, CONSTANS (CO) is the central gene activating the expression of the florigen FLOWERING LOCUS T (FT) in the leaves at the end of a long day. The circadian clock strongly regulates CO expression. However, to date, no evidence has been reported regarding a feedback loop from the photoperiod pathway back to the circadian clock. Using transcriptional networks, we have identified relevant network motifs regulating the interplay between the circadian clock and the photoperiod pathway. Gene expression, chromatin immunoprecipitation experiments, and phenotypic analysis allowed us to elucidate the role of CO over the circadian clock. Plants with altered CO expression showed a different internal clock period, measured by daily leaf rhythmic movements. We showed that CO upregulates the expression of key genes related to the circadian clock, such as CCA1, LHY, PRR5, and GI, at the end of a long day by binding to specific sites on their promoters. Moreover, a high number of PRR5-repressed target genes are upregulated by CO, and this could explain the phase transition promoted by CO. The CO-PRR5 complex interacts with the bZIP transcription factor HY5 and helps to localize the complex in the promoters of clock genes. Taken together, our results indicate that there may be a feedback loop in which CO communicates back to the circadian clock, providing seasonal information to the circadian system.

Quantitative RUBY reporter assay for gene regulation analysis.

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression.

OsbZIP1 regulates phosphorus uptake and nitrogen utilization, contributing to improved yield.

Increasing nutrient uptake and use efficiency in plants can contribute to improved crop yields and reduce the demand for fertilizers in crop production. In this study, we characterized a rice mutant, 88n which showed long roots under low nitrogen (N) or phosphorus (P) conditions. Low expression levels of N transporter genes were observed in 88n root, and total N concentration in 88n shoots were decreased, however, C concentrations and shoot dry weight in 88n were comparable to that in WT. Therefore, 88n showed high nitrogen utilization efficiency (NUtE). mRNA accumulation of Pi transporter genes was higher in 88n roots, and Pi concentration and uptake activity were higher in 88n than in WT. Therefore, 88n also showed high phosphorus uptake efficiency (PUpE). Molecular genetic analysis revealed that the causal gene of 88n phenotypes was OsbZIP1, a monocot-specific ortholog of the A. thaliana bZIP transcription factor HY5. Similar to the hy5 mutant, chlorophyll content in roots was decreased and root angle was shallower in 88n than in WT. Finally, we tested the yield of 88n in paddy fields over 3 years because 88n mutant plants showed higher PUpE and NUtE activity and different root architecture at the seedling stage. 88n showed large panicles and increased panicle weight/plant. Taken together, a mutation in OsbZIP1 could contribute to improved crop yields.

Advances in the endogenous and exogenous regulation of anthocyanins–the key to color change in eudicots

Anthocyanins are important water-soluble pigments that are widely found in plants and determine the color of plant organs, which possess significant health and economic value. The anthocyanin biosynthetic pathway is well established and some key regulators controlling this pathway have been identified in a variety of species. In recent years, microRNAs, transcription factors, and external factors, including light, temperature and hormones have emerged as central regulators of a variety of secondary metabolic responses, including anthocyanin metabolism, as both plant self-regulatory factors and environmental signals that stimulate plant development. Many discoveries have begun to suggest gene/signal-gene modules to reveal the signaling pathways involved in anthocyanin biosynthesis during fruit ripening and the links between specific genes. This paper reviews the internal and external factors and associated network mechanisms that regulate anthocyanin biosynthesis and accumulation in eudicots plants. miRNAs have different effects on the biosynthesis of anthocyanins in different species by binding to their target genes to form regulatory modules. The review also focuses on the conclusion that light signals (light quality, photoperiod, and light intensity) further regulate anthocyanin accumulation by binding to their corresponding photoreceptors (UVR8, CRYs, and PHYs) and affecting the binding and stabilization of the central regulators of the light pathway, such as COP1 and the transcription factor HY5. This article aims to provide a reference for future research on the regulation of anthocyanin by effective pathways, and to contribute to the selection of new varieties and the improvement of cultivation facilities.

Multifactorial in vivo regulation of the photoreceptor channelrhodopsin-1 abundance.

Oriented movement (phototaxis) is an efficient way to optimize light-driven processes and to avoid photodamage for motile algae. In Chlamydomonas the receptors for phototaxis are the channelrhodopsins ChR1 and ChR2. Both are directly light-gated, plasma membrane-localized cation channels. To optimally adjust its overall light-dependent responses, Chlamydomonas must tightly control the ChRs cellular abundance and integrate their activities into its general photoprotective network. How this is achieved is largely unknown. Here we show that the ChR1 protein level decreases upon illumination in a light-intensity and quality-dependent manner, whereas it is stable in prolonged darkness. Analysis of knockout strains of six major photoreceptors absorbing in the blue-violet range, which is most effective in evoking ChR1 degradation, revealed that only phototropin (PHOT) is involved. Notably, ChR2 degradation was normal in a ΔPHOT strain. Further, our results indicate that a COP1-SPA1 E3 ubiquitin ligase, the transcription factor Hy5 as well as changes in the cellular redox poise and cyclic nucleotide levels are additional components involved in this light acclimation response of Chlamydomonas. Our data highlight the presence of an adaptive framework connecting phototaxis with general photoprotective mechanisms via the use of overlapping signaling components already at the level of the primary photoreceptor.

Photosynthetic sucrose drives the lateral root clock in Arabidopsis seedlings

The development of plant roots is subject to control by light. Here, we show that, similar to monotonous root elongation, the periodic induction of lateral roots (LRs) depends on the activation by light of photomorphogenic and photosynthetic photoreceptors in the shoot in a hierarchical order. The prevailing belief is that the plant hormone auxin serves as a mobile signal transmitter, responsible for interorgan communication, including light-controlled shoot-to-root connections. Alternatively, it has been proposed that the transcription factor HY5 assumes the role as a mobile shoot-to-root signal transmitter. Here, we provide evidence that photosynthetic sucrose produced in the shoot acts as the long-distance signal carrier regulating the local, tryptophan-based biosynthesis of auxin in the LR generation zone of the primary root tip, where the LR clock controls the pace of LR initiation in an auxin-tunable manner. Synchronization of LR formation with primary root elongation allows the adjustment of overall root growth to the photosynthetic performance of the shoot and the maintenance of a constant LR density during light-dark changes in a variable light environment.

Growth and flavonol accumulation of Ginkgo biloba leaves affected by red and blue light

Ginkgo biloba L. is an economically important tree species in China rich in resources. Flavonoids in their leaves have a high medicinal value. Research on improving the flavonoid yield of G. biloba leaves by regulating environmental factors has been widely conducted. However, the effect of light quality on G. biloba growth and flavonoid accumulation is not clear, and its molecular mechanism has not been reported. In this study, one-year-old ginkgo seedlings were used as experimental material. Three treatments of red, mixed (red:blue = 1:1), and blue light were set up using LED lamps. Using white light as the control, differences of G. biloba growth and flavonol accumulation under different light qualities were explored. The results showed that under red, mixed, and blue light, the petiole of G. biloba elongated, the leaf area decreased, and the leaves tended to be slender, which was not conducive to the accumulation of leaves. The blue light significantly increased the height of G. biloba seedlings. The net photosynthetic rate under blue light was relatively lower, the transpiration rate was lower in the treatment groups, the stomatal conductance decreased significantly under mixed light, and the change in intercellular CO2 concentration was not significant. RNA sequencing (RNA-seq) combined with quantitative real-time polymerase chain reaction (qRT-PCR) showed that the transcription factors (TFs) HY5 (Gb_12012) and MYB (Gb_39081) and structural genes CHS (Gb_20355), F3H (Gb_29563), and FLS (Gb_14028, Gb_14029, and Gb_14030) positively regulated the biosynthesis of flavonols, whereas MYB-related TFs (Gb_27262 and Gb_34639) played a negative regulatory role. Combined with the existing research and the results of this experiment, we preliminarily speculated the mechanism of G. biloba response to red and blue light quality. After G. biloba leaves were stimulated by red and blue light, the expression of Gb_12012 changed the abundance of HY5 and affected the expression levels of downstream MYB TFs. MYB directly binds to the promoters of the structural genes CHS, F3H, and FLS to regulate its expression to promote (or inhibit) the biosynthesis of flavonols. Finally, Mixed and blue light significantly increased the flavonol content in G. biloba leaves, while red light was the opposite. Blue light significantly increased the flavone yield of G. biloba leaves per plant (increased by 0.76 times) and its antioxidant capacity. This lays a foundation for applying light quality in the cultivation of G. biloba for leaf use.

Pathway analysis of the proteomes of red, brown, and green macroalgae

Comparative proteomics provides insights into the biochemical pathways of macroalgae. One type of comparative proteomics is pathway analysis, which seeks to identify biological processes and cellular functions impacted by variations in groups of genes. Here we perform pathway analysis of macroalgae, including green (Ulva mutabilis and Ulva prolifera), red (Chondrus crispus, Gracilariopsis chorda and Porphyra umbilicalis), and brown (Ectocarpus siliculosus, Macrocystis pyrifera, Fucus vesiculosus, Saccharina japonica and Undaria pinnatifida) seaweeds. We chose these macroalgae because their whole genomes are publicly available. We determine gene function through mapping to orthologous gene groups in the KEGG database. Our analysis finds a core of approximately fifty pathways that are similar among groups, including central carbon metabolism (amino acid metabolism, nucleotide metabolism) and secondary metabolism (terpenoid biosynthesis, cofactors and vitamins, sulfur metabolism). We also determine numerous pathways that have differential orthologous gene group abundance among the three groups: central metabolic processes (carbon fixation, photosynthesis), secondary metabolism (fatty acid elongation, O-glycan biosynthesis), and also cellular processes (circadian rhythm, longevity regulation). We find both inter- and intra-group differences in the circadian rhythm pathway. For example, transcription factor HY5 is consistent in the red algae and absent from others, whereas, circadian clock associated 1 (CCA1) is present in 2 browns (E. siliculosus and S. japonica) and 2 reds (G. chorda and P. umbilicalis). In both of the green macroalgae and 2 reds (C. crispus and G. chorda) we identified a cryptochrome (CRY), which is a blue light receptor important in circadian rhythm. Overall, we find few orthologous gene groups in the circadian rhythm pathway to be present in the brown macroalgae except for pseudo-response regulator 7, which is consistent with a poorly understood “brown clock”.

Differential gene expression in chronically irradiated herbaceous species from the Chernobyl exclusion zone

Purpose Transcriptional activity of genes related to ionizing radiation responses in chronically irradiated plant populations at radioactively contaminated territories can be a cost-effective and precise approach for stress response evaluation. However, there are limits to studying non-model plants in field conditions. The work studies the transcriptional activity of candidate genes of adaptation to chronic radiation exposure in plant populations from radioactively contaminated territories of the Chernobyl. Materials and methods In this work, we studied plant species with different sensitivity to acute irradiation: Trifolium repens L., Taraxacum officinale Wigg., and Dactylis glomerata L., sampled in the Chernobyl exclusion zone. The differential expression of several candidate genes of adaptation to chronic radiation exposure in the leaves of these species was analyzed, including homologs of Arabidopsis thaliana genes SLAC1, APX1, GPX2, CAB1, NTRB, PP2-B11, RBOH-F, HY5, SnRK2.4, PDS1, CIPK20, SIP1, PIP1, TIP1. Results and conclusions All studied species were characterized by upregulation of the CAB1 homolog, encoding chlorophyll a/b binding protein, at radioactively contaminated plots. An increase in the expression of genes associated with water and hydrogen peroxide transport, intensity of photosynthesis, and stress responses (homolog of aquaporin TIP1 for T. repens; homologs of aquaporin PIP1 and transcription factor HY5 for D. glomerata; homolog of CBL-interacting serine/threonine protein kinase CIPK20 for T. officinale) was revealed. The methodological approach for studying gene expression in non-model plant species is described, which may allow large-scale screening studies of candidate genes in various plant species abundant in radioactively contaminated areas.

The UV-B-Induced Transcription Factor HY5 Regulated Anthocyanin Biosynthesis in Zanthoxylum bungeanum.

Pericarp color is an important economic characteristic of Zanthoxylum bungeanum. Anthocyanins are the main reason for the pericarp’s red appearance in Z. bungeanum. In this study, through the combined analysis of the metabolome and transcriptome, HY5, whose expression is highly correlated to changes in the anthocyanin content, was screened and identified. Under natural ripening conditions, the Z. bungeanum fruit gradually changed in color from green to red, while bagging resulted in the fruit maintaining its green color. After unbagging, the fruit gradually turned red, and the ZbHY5 expression and anthocyanin content increased. In addition, the leaves changed from green to red after exposure to UV-B radiation, and the ZbHY5 expression and anthocyanin content increased. The transient overexpression of ZbHY5 deepened the redness of the Z. bungeanum leaves and promoted the expression of ZbHY5 and ZbMYB113 as well as anthocyanin accumulation. Bimolecular fluorescence complementation (BIFC) showed that there was an interaction between ZbHY5 and ZbMYB113. These results revealed that under UV-B irradiation, ZbHY5 might regulate the expression levels of the structural genes related to anthocyanin biosynthesis through combination with ZbMYB113, thereby affecting anthocyanin accumulation. This finding provides useful insights for further studies focusing on UV-B-induced anthocyanin accumulation in Z. bungeanum.

The CRY2-COP1-HY5-BBX7/8 module regulates blue light-dependent cold acclimation in Arabidopsis.

Light and temperature are two key environmental factors that coordinately regulate plant growth and development. Although the mechanisms that integrate signaling mediated by cold and red light have been unraveled, the roles of the blue light photoreceptors cryptochromes in plant responses to cold remain unclear. In this study, we demonstrate that the CRYPTOCHROME2 (CRY2)-COP1-HY5-BBX7/8 module regulates blue light-dependent cold acclimation in Arabidopsis thaliana. We show that phosphorylated forms of CRY2 induced by blue light are stabilized by cold stress and that cold-stabilized CRY2 competes with the transcription factor HY5 to attenuate the HY5-COP1 interaction, thereby allowing HY5 to accumulate at cold temperatures. Furthermore, our data demonstrate that B-BOX DOMAIN PROTEIN7 (BBX7) and BBX8 function as direct HY5 targets that positively regulate freezing tolerance by modulating the expression of a set of cold-responsive genes, which mainly occurs independently of the C-repeat-binding factor pathway. Our study uncovers a mechanistic framework by which CRY2-mediated blue-light signaling enhances freezing tolerance, shedding light on the molecular mechanisms underlying the crosstalk between cold and light signaling pathways in plants.

Effect of LED Spectrum on the Quality and Nitrogen Metabolism of Lettuce Under Recycled Hydroponics.

Light quality optimization is an efficient method for improving the growth and quality of lettuce in plant factories. In this study, lettuce seedlings were illuminated under different light-emitting diode (LED) lights, namely, red-blue (RB), red-blue-green (RBG), red-blue-purple (RBP), and red-blue-far-red (RBF) LED lights, to investigate the effect of light quality on growth, quality, and nitrogen metabolism. The combination of 75% red and 25% blue light was set as the basic light source, and 20% of green, purple and far-red light were added to basic light source, respectively. All the treatments were set to 200 μmol m–2 s–1. Results showed that the fresh weight and dry weight of aboveground lettuce under RBG, RBP, and RBF treatments were significantly lower than those under the RB treatment because of the decrease in the effective photon flux density for chlorophyll absorption. The vitamin C content of the lettuce leaves was increased by about 23% with the addition of purple light. For nitrate reduction, the addition of green light significantly increased the nitrite content of the lettuce leaves. It also promoted the reduction from nitrite to ammonium through the activation of the nitrite reductase (NiR) expression and enzyme activity. The nitrate and ammonium content decreased with the addition of purple light because of the inhibited NR and NiR expression and enzyme activity. For nitrogen assimilation, individual (e.g., Asp, Glu, and Leu) and total amino acids were induced to increase by adding green, purple, and far-red light. The addition of light was hypothesized to have inhibited protein biosynthesis, thereby causing the accumulation of amino acids. Correlation analysis showed that the relative expression levels between HY5 and NR/NiR presented a significantly negative correlation. Transcription factor HY5 might mediate the regulation of light quality on nitrogen metabolism by inhibiting NR and NiR expressions. It might also exert a negative effect on nitrate reduction. Further studies via genome editing techniques on the identification of HY5 functions for nitrate assimilation will be valuable. Nevertheless, the results of this work enrich the understanding of the effect of light quality on nitrate metabolism at the level of gene expression and enzyme activity.

Effect of high-intensity light and UV-B on photosynthetic activity and the expression of certain light-responsive genes in A. thaliana phyA and phyB mutants

The effects of high-intensity light (HIL, 4 and 24 h) and UV-B (1 h) on the net photosynthesis rate, activity of photosystem II (PSII), content of photosynthetic pigments, anthocyanin and UV-absorbing pigments as well as the expression of certain light-responsive genes (HY5,CAB1) chalcone synthase (CHS) and main antioxidants enzyme genes (APX1, GPX and GR) in the leaves of phyB and phyA mutant A. thaliana plants were studied. Both UV-B and 4 and 24 h HIL decreased the PSII maximum quantum yield (Fv/Fm), PSII performance index (PIABS), photosynthesis and respiration rates in plants. Moreover, all stress treatments increased the dissipation of the absorbed energy (DI0/RC) as well as the flux of absorbed energy per RC (ABS/RC). The maximal changes in photosynthesis and chlorophyll fluorescence parameters were observed in the phyB mutant. The WT and the phyA mutant showed similar responses. In addition, the phyB mutant exhibited decreases in the expression of genes encoding enzyme CHS, the transcription factor HY5 and the antioxidant enzymes APX1 and GPX. One of the possible mechanisms protecting the photosynthetic apparatus from light excess or UV radiation is the elevated content of various pigments that can act as antioxidants or optical filters. We assume that the greater decrease in photosynthetic activity in the phyB mutant under HIL and UV-B conditions was due to the decreased content of carotenoids and UV-absorbing pigments in this mutant.

Arabidopsis downy mildew effector HaRxLL470 suppresses plant immunity by attenuating the DNA-binding activity of bZIP transcription factor HY5.

The oomycete pathogen Hyaloperonospora arabidopsidis delivers diverse effector proteins into host plant cells to suppress the plant's innate immunity. In this study, we investigate the mechanism of action of a conserved RxLR effector, HaRxLL470, in suppressing plant immunity. Genomic, molecular and biochemical analyses were performed to investigate the function of HaRxLL470 and the mechanism of the interaction between HaRxLL470 and the target host protein during H. arabidopsidis infection. We report that HaRxLL470 enhances plant susceptibility to H. arabidopsidis isolate Noco2 by interacting with the host photomorphogenesis regulator protein HY5. Our results demonstrate that HY5 is not only an important component in the regulation of light signalling, but also positively regulates host plant immunity against H. arabidopsidis by transcriptional activation of defense-related genes. We show that the interaction between HaRxLL470 and HY5 compromises the function of HY5 as a transcription factor by attenuating its DNA-binding activity. The present study demonstrates that HY5 positively regulates host plant defense against H. arabidopsidis whereas HaRxLL470, a conserved RxLR effector across oomycete pathogens, enhances pathogenicity by interacting with HY5 and suppressing transcriptional activation of defense-related genes.

ELONGATED HYPOCOTYL 5 mediates blue light-induced starch degradation in tomato.

Starch is the major storage carbohydrate in plants, and its metabolism in chloroplasts depends mainly on light. However, the mechanism through which photoreceptors regulate starch metabolism in chloroplasts is unclear. In this study, we found that the cryptochrome 1a (CRY1a)-mediated blue light signal is critical for regulating starch accumulation by inducing starch degradation through the transcription factor HY5 in chloroplasts in tomato. cry1a mutants and HY5-RNAi plants accumulated more starch and presented lower transcript levels of starch degradation-related genes in their leaves than wild-type plants. Blue light significantly induced the transcription of starch degradation-related genes in wild-type and CRY1a- or HY5-overexpressing plants but had little effect in cry1a and HY5-RNAi plants. Dual-luciferase assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation-qPCR revealed that HY5 could activate the starch degradation-related genes PWD, BAM1, BAM3, BAM8, MEX1, and DPE1 by directly binding to their promoters. Silencing of HY5 and these starch degradation-related genes in CRY1a-overexpressing plants led to increased accumulation of starch and decreased accumulation of soluble sugars. The findings presented here not only deepen our understanding of how light controls starch degradation and sugar accumulation but also allow us to explore potential targets for improving crop quality.

Anthocyanins from Ocimum sanctum L., a promising biomolecule for development of cost-effective and widely applicable pH indicator.

A novel cost-effective and widely applicable pH indicator was developed using anthocyanins extracted from the purple subtype of Ocimum sanctum L. and common lab filter paper. This pH indicator was successfully tested to monitor the pH of a wide range of buffers, solutions, irrigation water, and soil solution. Upon testing, the indicator displayed specific colors at corresponding pH ranges. Sucrose showed a stabilizing effect for the color of the extracted anthocyanins. Further, molecular analysis indicated that the leaves from the purple subtypes showed higher transcripts abundance for chalcone synthase, chalcone isomerase, anthocyanidin synthase, and dihydroflavonol 4-reductase than that of the green subtype. Similarly, transcription factors HY5 and a bHLH putatively involved in the biosynthesis of anthocyanins showed up-regulation in the purple subtype of O. sanctum.

Molecular-genetic mechanisms of regulation of dihydroflavonol reductase and transcription factor Hy5 by exogenous 5-aminolevulinic acid in winter rape plants

The effect of exogenous 5-aminolevulinic acid (ALA) on the activity of dihydroflavonol-4-reductase (DFR), the expression of the dfr gene and the hy5 gene of the transcription factor Hy5 and the light effect of different intensities in combination with the ALA action on the accumulation of anthocyanins in cotyledonous leaves of winter rape (Brassica napus L.) were studied. It was shown that the stimulation of the accumulation of anthocyanins under the exogenous ALA action at the molecular level was provided by increasing the expression level of the dfr and hy5 genes and the activity of the DFR enzyme. Increasing the light intensity from 40.5 to 66.2 μmol photons/m2·s enhanced the ability of plants to accumulate anthocyanins on average by 35 %. The ALA action at concentrations of 50, 100, 150 and 200 mg/L led to an additional increase in the accumulation of anthocyanins at the two used levels of illumination, and in a dose-dependent manner. The stimulating effect of ALA under high light intensity was much higher than in the case of lower illumination. Thus, the stimulation of the anthocyanin accumulation under illumination of 40.5 μmol photons/m2·s was 106 % when using 50 mg/L ALA, 165 % – when using 100 mg/L ALA, 222 % – in the case of 150 mg/L ALA and 350 % – under the action of 200 mg/L ALA compared with light control without of ALA treatment. At an illumination of 66.2 μmol photons/m2·s, these indicators were 164, 262, 359 and 583 % respectively. Thus, it was demonstrated that the stimulation of the accumulation of anthocyanins under the action of ALA in winter rape plants was due to its positive effect on the transcription of the dfr and hy5 genes at the molecular level.

Local HY5 Activity Mediates Hypocotyl Growth and Shoot-to-Root Communication

Plants optimize their growth in fluctuating environments using information acquired by different organs. This information is then transmitted through the rest of the plant using both short- and long-distance signals, including hormones and mobile proteins. Although a few of these signals have been characterized, long-distance signaling is not well understood in plants. Recently, the light-regulated transcription factor HY5 was reported to move from the shoot to the root to regulate root growth. We generated a cell-type specifically expressed HY5 fusion protein that could not be detected outside the tissue in which it was targeted. By expressing this DOF-HY5 protein in specific cell types of the hypocotyl, we showed that its local activity was sufficient to regulate hypocotyl growth. We also found that, although DOF-HY5 was expressed specifically in the shoot and not detected in the roots, it could rescue hy5 growth defects in primary roots but not in lateral roots. We therefore conclude that HY5 protein mobility is not required in the hypocotyl or for shoot-to-root communication. Our results indicate that a signal downstream of, or in parallel with, HY5 in the shoot is mobile and links shoot and root growth.

Rocket Science: The Effect of Spaceflight on Germination Physiology, Ageing, and Transcriptome of Eruca sativa Seeds.

In the ‘Rocket Science’ project, storage of Eruca sativa (salad rocket) seeds for six months on board the International Space Station resulted in delayed seedling establishment. Here we investigated the physiological and molecular mechanisms underpinning the spaceflight effects on dry seeds. We found that ‘Space’ seed germination vigor was reduced, and ageing sensitivity increased, but the spaceflight did not compromise seed viability and the development of normal seedlings. Comparative analysis of the transcriptomes (using RNAseq) in dry seeds and upon controlled artificial ageing treatment (CAAT) revealed differentially expressed genes (DEGs) associated with spaceflight and ageing. DEG categories enriched by spaceflight and CAAT included transcription and translation with reduced transcript abundances for 40S and 60S ribosomal subunit genes. Among the ‘spaceflight-up’ DEGs were heat shock proteins (HSPs), DNAJ-related chaperones, a heat shock factor (HSFA7a-like), and components of several DNA repair pathways (e.g., ATM, DNA ligase 1). The ‘response to radiation’ category was especially enriched in ‘spaceflight-up’ DEGs including HSPs, catalases, and the transcription factor HY5. The major finding from the physiological and transcriptome analysis is that spaceflight causes vigor loss and partial ageing during air-dry seed storage, for which space environmental factors and consequences for seed storage during spaceflights are discussed.

Chimeric Activators and Repressors Define HY5 Activity and Reveal a Light-Regulated Feedback Mechanism.

The first exposure to light marks a crucial transition in plant development. This transition relies on the transcription factor HY5 controlling a complex downstream growth program. Despite its importance, its function in transcription remains unclear. Previous studies have generated lists of thousands of potential target genes and competing models of HY5 transcription regulation. In this work, we carry out detailed phenotypic and molecular analysis of constitutive activator and repressor HY5 fusion proteins. Using this strategy, we were able to filter out large numbers of genes that are unlikely to be direct targets, allowing us to eliminate several proposed models of HY5's mechanism of action. We demonstrate that the primary activity of HY5 is promoting transcription and that this function relies on other, likely light-regulated, factors. In addition, this approach reveals a molecular feedback loop via the COP1/SPA E3 ubiquitin ligase complex, suggesting a mechanism that maintains low HY5 in the dark, primed for rapid accumulation to reprogram growth upon light exposure. Our strategy is broadly adaptable to the study of transcription factor activity. Lastly, we show that modulating this feedback loop can generate significant phenotypic diversity in both Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum).