What is going on inside of phytochrome B photobodies?

Plants exhibit an enormous phenotypic plasticity to adjust to changing environmental conditions. For this purpose, they have evolved mechanisms to detect and measure biotic and abiotic factors in their surroundings. Phytochrome B exhibits a dual function, since it serves as a photoreceptor for red and far-red light as well as a thermosensor. In 1999, it was first reported that phytochromes not only translocate into the nucleus but also form subnuclear foci upon irradiation by red light. It took more than 10 years until these phytochrome speckles received their name; these foci were coined photobodies to describe unique phytochrome-containing subnuclear domains that are regulated by light. Since their initial discovery, there has been much speculation about the significance and function of photobodies. Their presumed roles range from pure experimental artifacts to waste deposits or signaling hubs. In this review, we summarize the newest findings about the meaning of phyB photobodies for light and temperature signaling. Recent studies have established that phyB photobodies are formed by liquid-liquid phase separation via multivalent interactions and that they provide diverse functions as biochemical hotspots to regulate gene expression on multiple levels.

Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus.

HEMERA (HMR) is a nuclear and plastidial dual-targeted protein. While it functions in the nucleus as a transcriptional coactivator in phytochrome signaling to regulate a distinct set of light-responsive, growth-relevant genes, in plastids it is known as pTAC12, which associates with the plastid-encoded RNA polymerase, and is essential for inducing the plastomic photosynthetic genes and initiating chloroplast biogenesis. However, the mechanism of targeting HMR to the nucleus and plastids is still poorly understood. Here, we show that HMR can be directly imported into chloroplasts through a transit peptide residing in the N-terminal 50 amino acids. Upon cleavage of the transit peptide and additional proteolytic processing, mature HMR, which begins from Lys-58, retains its biochemical properties in phytochrome signaling. Unexpectedly, expression of mature HMR failed to rescue not only the plastidial but also the nuclear defects of the hmr mutant. This is because the predicted nuclear localization signals of HMR are nonfunctional, and therefore mature HMR is unable to accumulate in either plastids or the nucleus. Surprisingly, fusing the transit peptide of the small subunit of Rubisco with mature HMR rescues both its plastidial and nuclear localization and functions. These results, combined with the observation that the nuclear form of HMR has the same reduced molecular mass as plastidial HMR, support a retrograde protein translocation mechanism in which HMR is targeted first to plastids, processed to the mature form, and then relocated to the nucleus.

CYCLIN H;1 regulates drought stress responses and blue light-induced stomatal opening by inhibiting reactive oxygen species accumulation in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) CYCLIN-DEPENDENT KINASE Ds (CDKDs) phosphorylate the C-terminal domain of the largest subunit of RNA polymerase II. Arabidopsis CYCLIN H;1 (CYCH;1) interacts with and activates CDKDs; however, the physiological function of CYCH;1 has not been determined. Here, we report that CYCH;1, which is localized to the nucleus, positively regulates blue light-induced stomatal opening. Reduced-function cych;1 RNA interference (cych;1 RNAi) plants exhibited a drought tolerance phenotype. CYCH;1 is predominantly expressed in guard cells, and its expression was substantially down-regulated by dehydration. Transpiration of intact leaves was reduced in cych;1 RNAi plants compared with the wild-type control in light but not in darkness. CYCH;1 down-regulation impaired blue light-induced stomatal opening but did not affect guard cell development or abscisic acid-mediated stomatal closure. Microarray and real-time polymerase chain reaction analyses indicated that CYCH;1 did not regulate the expression of abscisic acid-responsive genes or light-induced stomatal opening signaling determinants, such as MYB60, MYB61, Hypersensitive to red and blue1, and Protein phosphatase7. CYCH;1 down-regulation induced the expression of redox homeostasis genes, such as LIPOXYGENASE3 (LOX3), LOX4, ARABIDOPSIS GLUTATHIONE PEROXIDASE 7 (ATGPX7), EARLY LIGHT-INDUCIBLE PROTEIN1 (ELIP1), and ELIP2, and increased hydrogen peroxide production in guard cells. Furthermore, loss-of-function mutations in CDKD;2 or CDKD;3 did not affect responsiveness to drought stress, suggesting that CYCH;1 regulates the drought stress response in a CDKD-independent manner. We propose that CYCH;1 regulates blue light-mediated stomatal opening by controlling reactive oxygen species homeostasis.

Transcriptional repression of GTL1 under water-deficit stress promotes anthocyanin biosynthesis to enhance drought tolerance.

The transcription factor GT2-LIKE 1 (GTL1) has been implicated in orchestrating a transcriptional network of diverse physiological, biochemical, and developmental processes. In response to water-limiting conditions, GTL1 is a negative regulator of stomatal development, but its potential rolein other water-deficit responses is unknown. We hypothesized that GTL1 regulates transcriptome changes associated with drought tolerance over leaf developmental stages. To test the hypothesis, gene expression was profiled by RNA-seq analysis in emerging and expanding leaves of wild-type and a drought-tolerant gtl1-4 knockout mutant under well-watered and water-deficit conditions. Our comparative analysis of genotype-treatment combinations within leaf developmental age identified 459 and 1073 differentially expressed genes in emerging and expanding leaves, respectively, as water-deficit responsive GTL1-regulated genes. Transcriptional profiling identified a potential role of GTL1 in two important pathways previously linked to drought tolerance: flavonoid and polyamine biosynthesis. In expanding leaves, negative regulation of GTL1 under water-deficit conditions promotes biosynthesis of flavonoids and anthocyanins that may contribute to drought tolerance. Quantification of polyamines did not support a role for GTL1 in these drought-responsive pathways, but this is likely due to the complex nature of polyamine synthesis and turnover. Our global transcriptome analysis suggests that transcriptional repression of GTL1 by water deficit allows plants to activate diverse pathways that collectively contribute to drought tolerance.

The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating stomatal density via transrepression of SDD1.

A goal of modern agriculture is to improve plant drought tolerance and production per amount of water used, referred to as water use efficiency (WUE). Although stomatal density has been linked to WUE, the causal molecular mechanisms have yet to be determined. Arabidopsis thaliana GT-2 LIKE 1 (GTL1) loss-of-function mutations result in increased water deficit tolerance and higher integrated WUE by reducing daytime transpiration without a demonstrable reduction in biomass accumulation. gtl1 plants had higher instantaneous WUE that was attributable to ~25% lower transpiration and stomatal conductance but equivalent CO(2) assimilation. Lower transpiration was associated with higher STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) expression and an ~25% reduction in abaxial stomatal density. GTL1 expression occurred in abaxial epidermal cells where the protein was localized to the nucleus, and its expression was downregulated by water stress. Chromatin immunoprecipitation analysis indicated that GTL1 interacts with a region of the SDD1 promoter that contains a GT3 box. An electrophoretic mobility shift assay was used to determine that the GT3 box is necessary for the interaction between GTL1 and the SDD1 promoter. These results establish that GTL1 negatively regulates WUE by modulating stomatal density via transrepression of SDD1.

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation.

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.

Sumoylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling.

SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. This paper presents evidence that the SUMO E3 ligase SIZ1 negatively regulates abscisic acid (ABA) signaling, which is dependent on the bZIP transcripton factor ABI5. Loss-of-function T-DNA insertion siz1-2 and siz1-3 mutations caused ABA hypersensitivity for seed germination arrest and seedling primary root growth inhibition. Furthermore, expression of genes that are ABA-responsive through ABI5-dependent signaling (e.g., RD29A, Rd29B, AtEm6, RAB18, ADH1) was hyperinduced by the hormone in siz1 seedlings. abi5-4 suppressed ABA hypersensitivity caused by siz1 (siz1-2 abi5-4), demonstrating an epistatic genetic interaction between SIZ1 and ABI5. A K391R substitution in ABI5 [ABI5(K391R)] blocked SIZ1-mediated sumoylation of the transcription factor in vitro and in Arabidopsis protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. In abi5-4 plants, ABI5(K391R) expression caused greater ABA hypersensitivity (gene expression, seed germination arrest and primary root growth inhibition) compared with ABI5 expression. Together, these results establish that SIZ1-dependent sumoylation of ABI5 attenuates ABA signaling. The double mutant siz1-2 afp-1 exhibited even greater ABA sensitivity than the single mutant siz1, suggesting that SIZ1 represses ABI5 signaling function independent of AFP1.

Anterograde signaling controls plastid transcription via sigma factors separately from nuclear photosynthesis genes.

Light initiates chloroplast biogenesis in Arabidopsis by eliminating PHYTOCHROME-INTERACTING transcription FACTORs (PIFs), which in turn de-represses nuclear photosynthesis genes, and synchronously, generates a nucleus-to-plastid (anterograde) signal that activates the plastid-encoded bacterial-type RNA polymerase (PEP) to transcribe plastid photosynthesis genes. However, the identity of the anterograde signal remains frustratingly elusive. The main challenge has been the difficulty to distinguish regulators from the plethora of necessary components for plastid transcription and other essential chloroplast functions, such as photosynthesis. Here, we show that the genome-wide induction of nuclear photosynthesis genes is insufficient to activate the PEP. PEP inhibition is imposed redundantly by multiple PIFs and requires PIF3's activator activity. Among the nuclear-encoded components of the PEP holoenzyme, we identify four light-inducible, PIF-repressed sigma factors as anterograde signals. Together, our results elucidate that light-dependent inhibition of PIFs activates plastid photosynthesis genes via sigma factors as anterograde signals in parallel with the induction of nuclear photosynthesis genes.

PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants.

The interplay between light receptors and PHYTOCHROME-INTERACTING FACTORs (PIFs) serves as a regulatory hub that perceives and integrates environmental cues into transcriptional networks of plants1,2. Although occupancy of the histone variant H2A.Z and acetylation of histone H3 have emerged as regulators of environmentally responsive gene networks, how these epigenomic features interface with PIF activity is poorly understood3-7. By taking advantage of rapid and reversible light-mediated manipulation of PIF7 subnuclear localization and phosphorylation, we simultaneously assayed the DNA-binding properties of PIF7, as well as its impact on chromatin dynamics genome wide. We found that PIFs act rapidly to reshape the H2A.Z and H3K9ac epigenetic landscape in response to a change in light quality. Furthermore, we discovered that PIFs achieve H2A.Z removal through direct interaction with EIN6 ENHANCER (EEN), the Arabidopsis thaliana homolog of the chromatin remodeling complex subunit INO80 Subunit 6 (Ies6). Thus, we describe a PIF-INO80 regulatory module that is an intermediate step for allowing plants to change their growth trajectory in response to environmental changes.

RCB initiates Arabidopsis thermomorphogenesis by stabilizing the thermoregulator PIF4 in the daytime.

Daytime warm temperature elicits thermomorphogenesis in Arabidopsis by stabilizing the central thermoregulator PHYTOCHROME INTERACTING transcription FACTOR 4 (PIF4), whose degradation is otherwise promoted by the photoreceptor and thermosensor phytochrome B. PIF4 stabilization in the light requires a transcriptional activator, HEMERA (HMR), and is abrogated when HMR's transactivation activity is impaired in hmr-22. Here, we report the identification of a hmr-22 suppressor mutant, rcb-101, which surprisingly carries an A275V mutation in REGULATOR OF CHLOROPLAST BIOGENESIS (RCB). rcb-101/hmr-22 restores thermoresponsive PIF4 accumulation and reverts the defects of hmr-22 in chloroplast biogenesis and photomorphogenesis. Strikingly, similar to hmr, the null rcb-10 mutant impedes PIF4 accumulation and thereby loses the warm-temperature response. rcb-101 rescues hmr-22 in an allele-specific manner. Consistently, RCB interacts directly with HMR. Together, these results unveil RCB as a novel temperature signaling component that functions collaboratively with HMR to initiate thermomorphogenesis by selectively stabilizing PIF4 in the daytime.

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B.

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3's transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms.

Quantitative Analysis of Photobodies.

Photobodies are membraneless subnuclear organelles that contain the red and far-red photoreceptors, phytochromes. Photobody biogenesis has been postulated to play important roles in early light signaling events. The size and number of photobodies are highly dynamic in response to the quality and quantity of light and correlated tightly with phytochrome-mediated seedling morphogenesis. Here, we provide a detailed protocol for characterization of the three-dimensional morphology of photobodies, including sample preparation, fluorescence microscopy, and image analysis. Although this method was developed initially for characterizing photobodies, it can be adopted to analyze other membraneless or membrane-bound subcellular organelles.

Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling.

Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.

A Ca2+/CaM-regulated transcriptional switch modulates stomatal development in response to water deficit.

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

NCP activates chloroplast transcription by controlling phytochrome-dependent dual nuclear and plastidial switches.

Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.

Growth of Arabidopsis thaliana and Eutrema salsugineum in a closed growing system designed for quantification of plant water use.

The identification of genetic determinants for water-use efficiency (WUE) and their incorporation into crop plants is critical as world water resources are predicted to become less stable over the coming decades. However, quantification of WUE in small model species such as Arabidopsis is difficult because of low plant water loss relative to root zone evaporation. Furthermore, measurements of long-term WUE are labor-intensive and time-consuming. A novel high-throughput closed-container growing system for measuring plant WUE is described. The system eliminates nearly all water loss from the media and does not require irrigation throughout the duration of a typical experiment. Using the model species Arabidopsis thaliana and Eutrema salsugineum, it was confirmed that under growth chamber conditions, this system: (1) eliminates the need for irrigation for as much as 30 days with media water content remaining above 80% full capacity; (2) allows for quantification of WUE in plants with a leaf area as small as ca. 20 cm(2); (3) does not inhibit plant growth; and (4) does not alter media conditions outside of an acceptable range for these species. The growing system provides an efficient high-throughput system for quantifying plant water loss and WUE.

PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis.

Plants react to seasonal change in day length through altering physiology and development. Factors that function to harmonize growth with photoperiod are poorly understood. Here we characterize a new protein that associates with both circadian clock and photoreceptor components, named PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1). pch1 seedlings have overly elongated hypocotyls specifically under short days while constitutive expression of PCH1 shortens hypocotyls independent of day length. PCH1 peaks at dusk, binds phytochrome B (phyB) in a red light-dependent manner, and co-localizes with phyB into photobodies. PCH1 is necessary and sufficient to promote the biogenesis of large photobodies to maintain an active phyB pool after light exposure, potentiating red-light signaling and prolonging memory of prior illumination. Manipulating PCH1 alters PHYTOCHROME INTERACTING FACTOR 4 levels and regulates light-responsive gene expression. Thus, PCH1 is a new factor that regulates photoperiod-responsive growth by integrating the clock with light perception pathways through modulating daily phyB-signaling.

Poplar GTL1 is a Ca2+/calmodulin-binding transcription factor that functions in plant water use efficiency and drought tolerance.

Diminishing global fresh water availability has focused research to elucidate mechanisms of water use in poplar, an economically important species. A GT-2 family trihelix transcription factor that is a determinant of water use efficiency (WUE), PtaGTL1 (GT-2 like 1), was identified in Populus tremula × P. alba (clone 717-IB4). Like other GT-2 family members, PtaGTL1 contains both N- and C-terminal trihelix DNA binding domains. PtaGTL1 expression, driven by the Arabidopsis thaliana AtGTL1 promoter, suppressed the higher WUE and drought tolerance phenotypes of an Arabidopsis GTL1 loss-of-function mutation (gtl1-4). Genetic suppression of gtl1-4 was associated with increased stomatal density due to repression of Arabidopsis STOMATAL DENSITY AND DISTRIBUTION1 (AtSDD1), a negative regulator of stomatal development. Electrophoretic mobility shift assays (EMSA) indicated that a PtaGTL1 C-terminal DNA trihelix binding fragment (PtaGTL1-C) interacted with an AtSDD1 promoter fragment containing the GT3 box (GGTAAA), and this GT3 box was necessary for binding. PtaGTL1-C also interacted with a PtaSDD1 promoter fragment via the GT2 box (GGTAAT). PtaSDD1 encodes a protein with 60% primary sequence identity with AtSDD1. In vitro molecular interaction assays were used to determine that Ca(2+)-loaded calmodulin (CaM) binds to PtaGTL1-C, which was predicted to have a CaM-interaction domain in the first helix of the C-terminal trihelix DNA binding domain. These results indicate that, in Arabidopsis and poplar, GTL1 and SDD1 are fundamental components of stomatal lineage. In addition, PtaGTL1 is a Ca(2+)-CaM binding protein, which infers a mechanism by which environmental stimuli can induce Ca(2+) signatures that would modulate stomatal development and regulate plant water use.

Regulation of stomatal density by the GTL1 transcription factor for improving water use efficiency.

A stoma (pl. stomata) is the pore formed by two guard cells found predominantly in the leaf epidermis. Plants control stomatal aperture (opening and closing) and/or number (density) to regulate carbon dioxide (CO2) uptake and water loss (transpiration), which is necessary to optimize plant growth, development, and fitness in response to various environmental conditions. Recently, we identified Arabidopsis GT2-LIKE 1 (GTL1) as a transcriptional repressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), a negative regulator of stomatal density. GTL1 directly interacts with the SDD1 promoter regulating stomatal density, transpiration, and water use efficiency (WUE). Here we discuss potential GTL1 orthologs in other plant species.