A synchronized, large-scale field experiment using Arabidopsis thaliana reveals the significance of the UV-B photoreceptor UVR8 under natural conditions.

This study determines the functional role of the plant ultraviolet-B radiation (UV-B) photoreceptor, UV RESISTANCE LOCUS 8 (UVR8) under natural conditions using a large-scale 'synchronized-genetic-perturbation-field-experiment'. Laboratory experiments have demonstrated a role for UVR8 in UV-B responses but do not reflect the complexity of outdoor conditions where 'genotype × environment' interactions can mask laboratory-observed responses. Arabidopsis thaliana knockout mutant, uvr8-7, and the corresponding Wassilewskija wild type, were sown outdoors on the same date at 21 locations across Europe, ranging from 39°N to 67°N latitude. Growth and climatic data were monitored until bolting. At the onset of bolting, rosette size, dry weight, and phenolics and glucosinolates were quantified. The uvr8-7 mutant developed a larger rosette and contained less kaempferol glycosides, quercetin glycosides and hydroxycinnamic acid derivatives than the wild type across all locations, demonstrating a role for UVR8 under field conditions. UV effects on rosette size and kaempferol glycoside content were UVR8 dependent, but independent of latitude. In contrast, differences between wild type and uvr8-7 in total quercetin glycosides, and the quercetin-to-kaempferol ratio decreased with increasing latitude, that is, a more variable UV response. Thus, the large-scale synchronized approach applied demonstrates a location-dependent functional role of UVR8 under natural conditions.

Phytochrome B phosphorylation expanded: site-specific kinases are identified.

The phytochrome B (phyB) photoreceptor is a key participant in red and far-red light sensing, playing a dominant role in many developmental and growth responses throughout the whole life of plants. Accordingly, phyB governs diverse signaling pathways, and although our knowledge about these pathways is constantly expanding, our view about their fine-tuning is still rudimentary. Phosphorylation of phyB is one of the relevant regulatory mechanisms, and - despite the expansion of the available methodology - it is still not easy to examine. Phosphorylated phytochromes have been detected using various techniques for decades, but the first phosphorylated phyB residues were only identified in 2013. Since then, concentrated attention has been turned toward the functional role of post-translational modifications in phyB signaling. Very recently in 2023, the first kinases that phosphorylate phyB were identified. These discoveries opened up new research avenues, especially by connecting diverse environmental impacts to light signaling and helping to explain some long-term unsolved problems such as the co-action of Ca2+ and phyB signaling. This review summarizes our recent views about the roles of the identified phosphorylated phyB residues, what we know about the enzymes that modulate the phospho-state of phyB, and how these recent discoveries impact future investigations.

A transcriptional complex of FtMYB102 and FtbHLH4 coordinately regulates the accumulation of rutin in Fagopyrum tataricum.

Tartary buckwheat is rich in flavonoids, which not only play an important role in the plant-environment interaction, but are also beneficial to human health. Rutin is a therapeutic flavonol which is massively accumulated in Tartary buckwheat. It has been demonstrated that transcription factors control rutin biosynthesis. However, the transcriptional regulatory network of rutin is not fully clear. In this study, through transcriptome and target metabolomics, we validated the role of FtMYB102 and FtbHLH4 TFs at the different developmental stages of Tartary buckwheat. The elevated accumulation of rutin in the sprout appears to be closely associated with the expression of FtMYB102 and FtbHLH4. Yeast two-hybrid, transient luciferase activity and co-immunoprecipitation demonstrated that FtMYB102 and FtbHLH4 can interact and form a transcriptional complex. Moreover, yeast one-hybrid showed that both FtMYB102 and FtbHLH4 directly bind to the promoter of chalcone isomerase (CHI), and they can coordinately induce CHI expression as shown by transient luciferase activity assay. Finally, we transferred FtMYB102 and FtbHLH4 into the hairy roots of Tartary buckwheat and found that they both can promote the accumulation of rutin. Our results indicate that FtMYB102 and FtbHLH4 can form a transcriptional complex by inducing CHI expression to coordinately promote the accumulation of rutin.

SUMOylation of different targets fine-tunes phytochrome signaling.

Plants monitor their surrounding ambient light environment by specialized photoreceptor proteins. Among them, phytochromes monitor red and far-red light. These molecules perceive photons, undergo a conformational change, and regulate diverse light signaling pathways, resulting in the mediation of key developmental and growth responses throughout the whole life of plants. Posttranslational modifications of the photoreceptors and their signaling partners may modify their function. For example, the regulatory role of phosphorylation has been investigated for decades by using different methodological approaches. In the past few years, a set of studies revealed that ubiquitin-like short protein molecules, called small ubiquitin-like modifiers (SUMOs) are attached reversibly to different members of phytochrome signaling pathways, including phytochrome B, the dominant receptor of red light signaling. Furthermore, SUMO attachment modifies the action of the target proteins, leading to altered light signaling and photomorphogenesis. This review summarizes recent results regarding SUMOylation of various target proteins, the regulation of their SUMOylation level, and the physiological consequences of SUMO attachment. Potential future research directions are also discussed.

Uncovering a novel function of the CCR4-NOT complex in phytochrome A-mediated light signalling in plants.

Phytochromes are photoreceptors regulating growth and development in plants. Using the model plant Arabidopsis, we identified a novel signalling pathway downstream of the far-red light-sensing phytochrome, phyA, that depends on the highly conserved CCR4-NOT complex. CCR4-NOT is integral to RNA metabolism in yeast and animals, but its function in plants is largely unknown. NOT9B, an Arabidopsis homologue of human CNOT9, is a component of the CCR4-NOT complex, and acts as negative regulator of phyA-specific light signalling when bound to NOT1, the scaffold protein of the complex. Light-activated phyA interacts with and displaces NOT9B from NOT1, suggesting a potential mechanism for light signalling through CCR4-NOT. ARGONAUTE 1 and proteins involved in splicing associate with NOT9B and we show that NOT9B is required for specific phyA-dependent alternative splicing events. Furthermore, association with nuclear localised ARGONAUTE 1 raises the possibility that NOT9B and CCR4-NOT are involved in phyA-modulated gene expression.

Place a seedling on a windowsill, and soon you will notice the fragile stem bending towards the glass to soak in the sun and optimize its growth. Plants can 'sense' light thanks to specialized photoreceptor molecules: for instance, the phytochrome A is responsible for detecting weak and 'far-red' light from the very edge of the visible spectrum. Once the phytochrome has been activated, this message is relayed to the rest of the plant through an intricate process that requires other molecules. The CCR4-NOT protein complex is vital for all plants, animals and fungi, suggesting that it was already present in early life forms. Here, Schwenk et al. examine whether CCR4-NOT could have acquired a new role in plants to help them respond to far-red light. Scanning the genetic information of the plant model Arabidopsis thaliana revealed that the gene encoding the NOT9 subunit of CCR4-NOT had been duplicated in plants during evolution. NOT9B, the protein that the new copy codes for, has a docking site that can attach to both phytochrome A and CCR4-NOT. When NOT9B binds phytochrome A, it is released from the CCR4-NOT complex: this could trigger a cascade of reactions that ultimately changes how A. thaliana responds to far-red light. Plants that had not enough or too much NOT9B were respectively more or less responsive to that type of light, showing that the duplication of the gene coding for this subunit had helped plants respond to certain types of light. The findings by Schwenk et al. illustrate how existing structures can be repurposed during evolution to carry new roles. They also provide a deeper understanding of how plants optimize their growth, a useful piece of information in a world where most people rely on crops as their main source of nutrients.

SUMOylation of PHYTOCHROME INTERACTING FACTOR 3 promotes photomorphogenesis in Arabidopsis thaliana.

In Arabidopsis thaliana, phytochrome B (phyB) is the dominant receptor of photomorphogenic development under red light. Phytochrome B interacts with a set of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). The interaction between PIF3 and photoactivated phyB leads to the rapid phosphorylation and degradation of PIF3 and also to the degradation of phyB, events which are required for proper photomorphogenesis. Here we report that PIF3 is SUMOylated at the Lys13 (K13) residue and that we could detect this posttranslational modification in a heterologous experimental system and also in planta. We also found that the SUMO acceptor site mutant PIF3(K13R) binds more strongly to the target promoters than its SUMOylated, wild-type counterpart. Seedlings expressing PIF3(K13R) show an elongated hypocotyl response, elevated photoprotection and higher transcriptional induction of red-light responsive genes compared with plantlets expressing wild-type PIF3. These observations are supported by the lower level of phyB in plants which possess only PIF3(K13R), indicating that SUMOylation of PIF3 also alters photomorphogenesis via the regulation of phyB levels. In conclusion, whereas SUMOylation is generally connected to different stress responses, it also fine-tunes light signalling by reducing the biological activity of PIF3, thus promoting photomorphogenesis.

Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling.

Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants' light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr-Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N-terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling.

Light Triggers the miRNA-Biogenetic Inconsistency for De-etiolated Seedling Survivability in Arabidopsis thaliana.

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1, SERRATE, and HYPONASTIC LEAVES 1, whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels. However, the levels of most miRNAs do not notably increase in response to light. To reconcile this inconsistency, we demonstrated that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, these data suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.

A Deep Learning-Based Approach for High-Throughput Hypocotyl Phenotyping.

Hypocotyl length determination is a widely used method to phenotype young seedlings. The measurement itself has advanced from using rulers and millimeter papers to assessing digitized images but remains a labor-intensive, monotonous, and time-consuming procedure. To make high-throughput plant phenotyping possible, we developed a deep-learning-based approach to simplify and accelerate this method. Our pipeline does not require a specialized imaging system but works well with low-quality images produced with a simple flatbed scanner or a smartphone camera. Moreover, it is easily adaptable for a diverse range of datasets not restricted to Arabidopsis (Arabidopsis thaliana). Furthermore, we show that the accuracy of the method reaches human performance. We not only provide the full code at https://github.com/biomag-lab/hypocotyl-UNet, but also give detailed instructions on how the algorithm can be trained with custom data, tailoring it for the requirements and imaging setup of the user.

Detection of Phytochrome Phosphorylation in Plants.

Posttranslational modification (PTM) of proteins occurs during or after translation and in most cases means covalent binding of a functional group to certain amino acid side chains. Among PTMs, phosphorylation is extensively studied for decades. During phosphorylation, a phosphate group is added to the target residue that is dominantly serine, threonine, and tyrosine in eukaryotes. The phosphate group attachment is catalyzed by kinases, whereas the removal of phosphate (dephosphorylation) is performed by phosphatases. Phosphorylation of phytochrome photoreceptors alters light signaling in multiple ways, thus the examination of this PTM is an expanding aspect of light signaling research. Although this chapter presents methods for detecting phosphorylated phytochrome B molecules, it can be applied on other phytochrome species. The first presented protocol of this chapter shows how the phosphorylation state of phytochrome photoreceptors can be monitored in a modified polyacrylamide gel electrophoresis system. The second protocol describes in detail how phosphorylated amino acids of a target molecule can be identified using mass spectrometry analysis.

Detection of SUMOylated Phytochromes in Plants.

Posttranslational modifications (PTMs) happen after or during protein translation. Small Ubiquitin-like Modifier (SUMO) proteins are covalently attached to certain lysine residues of the target proteins to modify their activity, stability, or localization. This process is called SUMOylation, which is a reversible PTM: SUMO protease enzymes can cleave SUMOs off the target protein backbone. Although many ubiquitinated proteins are targeted for degradation, SUMOylation does not necessary lead to the degradation of the modified protein but lead to the regulation of various physiological responses. SUMOylation of the examined protein cannot simply be monitored by immunoblotting techniques performed on total protein extracts, due to the SUMO-specific signals derived from other modified molecules. Furthermore, the fact that only a limited fraction of the target protein pool is SUMOylated makes the detection of SUMOylated proteins challenging. This protocol shows how SUMOylated phytochrome B (phyB) molecules can be detected using homologous and heterologous experimental systems in planta.

Differential UVR8 Signal across the Stem Controls UV-B-Induced Inflorescence Phototropism.

In the course of evolution, plants have developed mechanisms that orient their organs toward the incoming light. At the seedling stage, positive phototropism is mainly regulated by phototropin photoreceptors in blue and UV wavelengths. Contrasting with this, we report that UV RESISTANCE LOCUS8 (UVR8) serves as the predominant photoreceptor of UV-B-induced phototropic responses in Arabidopsis (Arabidopsis thaliana) inflorescence stems. We examined the molecular mechanisms underlying this response and our findings support the Blaauw theory (Blaauw, 1919), suggesting rapid differential growth through unilateral photomorphogenic growth inhibition. UVR8-dependent UV-B light perception occurs mainly in the epidermis and cortex, but deeper tissues such as endodermis can also contribute. Within stems, a spatial difference of UVR8 signal causes a transcript and protein increase of transcription factors ELONGATED HYPOCOTYL5 (HY5) and its homolog HY5 HOMOLOG at the UV-B-exposed side. The irradiated side shows (1) strong activation of flavonoid synthesis genes and flavonoid accumulation; (2) increased gibberellin (GA)2-oxidase expression, diminished GA1 levels, and accumulation of the DELLA protein REPRESSOR OF GA1; and (3) increased expression of the auxin transport regulator PINOID, contributing to diminished auxin signaling. Together, the data suggest a mechanism of phototropin-independent inflorescence phototropism through multiple, locally UVR8-regulated hormone pathways.

UVR8-dependent reporters reveal spatial characteristics of signal spreading in plant tissues.

The UV Resistance Locus 8 (UVR8) photoreceptor controls UV-B mediated photomorphogenesis in Arabidopsis. The aim of this work is to collect and characterize different molecular reporters of photomorphogenic UV-B responses. Browsing available transcriptome databases, we identified sets of genes responding specifically to this radiation and are controlled by pathways initiated from the UVR8 photoreceptor. We tested the transcriptional changes of several reporters and found that they are regulated differently in different parts of the plant. Our experimental system led us to conclude that the examined genes are not controlled by light piping of UV-B from the shoot to the root or signalling molecules which may travel between different parts of the plant body but by local UVR8 signalling. The initiation of these universal signalling steps can be the induction of Elongated Hypocotyl 5 (HY5) and its homologue, HYH transcription factors. We found that their transcript and protein accumulation strictly depends on UVR8 and happens in a tissue autonomous manner. Whereas HY5 accumulation correlates well with the UVR8 signal across cell layers, the induction of flavonoids depends on both UVR8 signal and a yet to be identified tissue-dependent or developmental determinant.

Expression of the UVR8 photoreceptor in different tissues reveals tissue-autonomous features of UV-B signalling.

The Arabidopsis UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) orchestrates the expression of hundreds of genes, many of which can be associated with UV-B tolerance. UV-B does not efficiently penetrate into tissues, yet UV-B regulates complex growth and developmental responses. To unravel to what extent and how UVR8 located in different tissues contributes to UV-B-induced responses, we expressed UVR8 fused to the YELLOW FLUORESCENT PROTEIN (YFP) under the control of tissue-specific promoters in a uvr8 null mutant background. We show that (1) UVR8 localized in the epidermis plays a major role in regulating cotyledon expansion, and (2) expression of UVR8 in the mesophyll is important to protect adult plants from the damaging effects of UV-B. We found that UV-B induces transcription of selected genes, including the key transcriptional regulator ELONGATED HYPOCOTYL 5 (HY5), only in tissues that express UVR8. Thus, we suggest that tissue-autonomous and simultaneous UVR8 signalling in different tissues mediates, at least partly, developmental and defence responses to UV-B.

New insights of red light-induced development.

The red/far-red light absorbing photoreceptors phytochromes regulate development and growth and thus play an essential role in optimizing adaptation of the sessile plants to the ever-changing environment. Our understanding of how absorption of a red/far-red photon by phytochromes initiates/modifies diverse physiological responses has been steadily improving. Research performed in the last 5 years has been especially productive and led to significant conceptual changes about the mode of action of these photoreceptors. In this review, we focus on the phytochrome B photoreceptor, the major phytochrome species active in light-grown plants. We discuss how its light-independent inactivation (termed dark/thermal reversion), post-translational modification, including ubiquitination, phosphorylation and sumoylation, as well as heterodimerization with other phytochrome species modify red light-controlled physiological responses. Finally, we discuss how photobiological properties of phytochrome B enable this photoreceptor to function also as a thermosensor.

Characterization of photomorphogenic responses and signaling cascades controlled by phytochrome-A expressed in different tissues.

The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photomorphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5:HY5-GFP and Pro35S:CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion.

SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana.

The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyB(Lys996Arg)-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases.

Molecular mechanisms for mediating light-dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors.

The photoreceptors phytochromes monitor the red/far-red part of the spectrum, exist in the biologically active Pfr (far-red absorbing) or inactive Pr (red absorbing) forms, and function as red/far-red light-regulated molecular switches to modulate plant development and growth. Phytochromes are synthesized in the cytoplasm, and light induces translocation of the Pfr conformer into the nucleus. Nuclear import of phytochromes is a highly regulated process and is fine-tuned by the quality and quantity of light. It appears that phytochrome A (phyA) and phytochrome B (phyB) do not possess active endogenous nuclear import signals (NLSs), thus light-induced translocation of these photoreceptors into the nucleus requires direct protein­protein interactions with their NLS-containing signaling partners. Sub-cellular partitioning of the various phytochrome species is mediated by different molecular machineries. Translocation of phyA into the nucleus is promoted by FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL), but the identity of nuclear transport facilitators mediating the import of phyB-E into the nucleus remains elusive. Phytochromes localized in the nucleus are associated with specific protein complexes, termed photobodies. The size and distribution of these structures are regulated by the intensity and duration of irradiation, and circumstantial evidence indicates that they are involved in fine-tuning phytochrome signaling.

phyA-GFP is spectroscopically and photochemically similar to phyA and comprises both its native types, phyA' and phyA''.

Low-temperature fluorescence investigations of phyA-GFP used in experiments on its nuclear-cytoplasmic partitioning were carried out. In etiolated hypocotyls of phyA-deficient Arabidopsis thaliana expressing phyA-GFP, it was found that it is similar to phyA in spectroscopic parameters with both its native types, phyA' and phyA'', present and their ratio shifted towards phyA'. In transgenic tobacco hypocotyls, native phyA and rice phyA-GFP were also identical to phyA in the wild type whereas phyA-GFP belonged primarily to the phyA' type. Finally, truncated oat Δ6-12 phyA-GFP expressed in phyA-deficient Arabidopsis was represented by the phyA' type in contrast to full-length oat phyA-GFP with an approximately equal proportion of the two phyA types. This correlates with a previous observation that Δ6-12 phyA-GFP can form only numerous tiny subnuclear speckles while its wild-type counterpart can also localize into bigger and fewer subnuclear protein complexes. Thus, phyA-GFP is spectroscopically and photochemically similar or identical to the native phyA, suggesting that the GFP tag does not affect the chromophore. phyA-GFP comprises phyA'-GFP and phyA''-GFP, suggesting that both of them are potential participants in nuclear-cytoplasmic partitioning, which may contribute to its complexity.