Association mechanism between Arabidopsis immune coreceptor BAK1 and Pseudomonas syringae effector HopF2.

Brassinosteroid activated kinase 1 (BAK1) is a cell-surface coreceptor which plays multiple roles in innate immunity of plants. HopF2 is an effector secreted by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 into Arabidopsis and suppresses host immune system through interaction with BAK1 as well as its downstream kinase MKK5. The association mechanism of HopF2 to BAK1 remains unclear, which prohibits our understanding and subsequent interfering of their interaction for pathogen management. Herein, we found the kinase domain of BAK1 (BAK1-KD) is sufficient for HopF2 association. With a combination of hydrogen/deuterium exchange mass spectrometry and mutational assays, we found a region of BAK1-KD N-lobe and a region of HopF2 head subdomain are critical for intermolecular interaction, which is also supported by unbiased protein-protein docking with ClusPro and kinase activity assay. Collectively, this research presents the interaction mechanism between Arabidopsis BAK1 and P. syringae HopF2, which could pave the way for bactericide development that blocking the functioning of HopF2 toward BAK1.

Plants distinguish different photoperiods to independently control seasonal flowering and growth.

Plants measure daylength (photoperiod) to regulate seasonal growth and flowering. Photoperiodic flowering has been well studied, but less is known about photoperiodic growth. By using a mutant with defects in photoperiodic growth, we identified a seasonal growth regulation pathway that functions in long days in parallel to the canonical long-day photoperiod flowering mechanism. This is achieved by using distinct mechanisms to detect different photoperiods: The flowering pathway measures photoperiod as the duration of light intensity, whereas the growth pathway measures photoperiod as the duration of photosynthetic activity (photosynthetic period). Plants can then independently control expression of genes required for flowering or growth. This demonstrates that seasonal flowering and growth are dissociable, allowing them to be coordinated independently across seasons.

FLOWERING LOCUS T mediates photo-thermal timing of inflorescence meristem arrest in Arabidopsis thaliana.

Plants integrate environmental information into their developmental program throughout their lifetime. Light and temperature are particularly critical cues for plants to correctly time developmental transitions. Here, we investigated the role of photo-thermal cues in the regulation of the end-of-flowering developmental transition in the model plant Arabidopsis (Arabidopsis thaliana). We found that increased day length and higher temperature during flowering promote earlier inflorescence arrest by accelerating the rate at which the inflorescence meristem (IM) initiates floral primordia. Specifically, we show that plants arrest at a photo-thermal threshold and demonstrate that this photo-thermally mediated arrest is mediated by the floral integrator FLOWERING LOCUS T (FT), a known activator of flowering. FT expression increased over the duration of flowering, peaking during IM arrest, and we show that this is necessary and sufficient for photo-thermally induced arrest. Our data demonstrate the role of light and temperature, through FT, as key regulators of end-of-flowering. Overall, our results have important implications for understanding and modulating the flowering duration of crop species in changing light and temperature conditions in a warming global climate.

HDF1, a novel flowering time regulator identified in a mutant suppressing sensitivity to red light reduced 1 early flowering.

Arabidopsis SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) delays the transition from vegetative to reproductive development in noninductive conditions. A second-site suppressor screen for novel genes that overcome early flowering of srr1-1 identified a range of suppressor of srr1-1 mutants flowering later than srr1-1 in short photoperiods. Here, we focus on mutants flowering with leaf numbers intermediate between srr1-1 and Col. Ssm67 overcomes srr1-1 early flowering independently of day-length and ambient temperature. Full-genome sequencing and linkage mapping identified a causative SNP in a gene encoding a Haloacid dehalogenase superfamily protein, named HAD-FAMILY REGULATOR OF DEVELOPMENT AND FLOWERING 1 (HDF1). Both, ssm67 and hdf1-1 show increased levels of FLC, indicating that HDF1 is a novel regulator of this floral repressor. HDF1 regulates flowering largely independent of SRR1, as the effect is visible in srr1-1 and in Col, but full activity on FLC may require SRR1. Furthermore, srr1-1 has a delayed leaf initiation rate that is dependent on HDF1, suggesting that SRR1 and HDF1 act together in leaf initiation. Another mutant flowering intermediate between srr1-1 and wt, ssm15, was identified as a new allele of ARABIDOPSIS SUMO PROTEASE 1, previously implicated in the regulation of FLC stability.

Insights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C.

Non-specific phospholipase C (NPC) hydrolyzes major membrane phospholipids to release diacylglycerol (DAG), a potent lipid-derived messenger regulating cell functions. Despite extensive studies on NPCs reveal their fundamental roles in plant growth and development, the mechanistic understanding of phospholipid-hydrolyzing by NPCs, remains largely unknown. Here we report the crystal structure of Arabidopsis NPC4 at a resolution of 2.1 Å. NPC4 is divided into a phosphoesterase domain (PD) and a C-terminal domain (CTD), and is structurally distinct from other characterized phospholipases. The previously uncharacterized CTD is indispensable for the full activity of NPC4. Mechanistically, CTD contributes NPC4 activity mainly via CTDα1-PD interaction, which ultimately stabilizes the catalytic pocket in PD. Together with a series of structure-guided biochemical studies, our work elucidates the structural basis and provides molecular mechanism of phospholipid hydrolysis by NPC4, and adds new insights into the members of phospholipase family.

Effects of weak static magnetic fields on the development of seedlings of Arabidopsis thaliana.

To study magnetoreception of Arabidopsis thaliana, we analysed several developmental responses including cryptochrome-independent seed germination and the phytochrome- and cryptochrome-dependent hypocotyl elongation and photo-accumulation of anthocyanins and chlorophylls in weak static magnetic fields ranging from near null to 122 µT. A field of 50 µT accelerated seed germination by about 20 h relative to samples maintained in a near-null field. The double mutant, cry1cry2, lacking cryptochromes 1 and 2 displayed the same magnetic field-induced germination acceleration under blue light as the wild-type strain. Magnetic field-induced germination acceleration was masked in the presence of exogenous sucrose. Stimulus-response curves for hypocotyl elongation in a range between near-null to 122 µT indicated maxima near 9 and 60 µT for the wild-type strain as well as mutant cry1cry2. The photo-accumulation of anthocyanins and chlorophylls could be effectively modulated by magnetic fields in the presence of low-irradiance red and blue light, respectively. The findings indicate that Arabidopsis thaliana possesses light-independent mechanisms of magnetic field reception, which remain presently unidentified. Our results are in better agreement with predictions of the level crossing mechanism (LCM) of magnetoreception rather than those of the cryptochrome-associated radical-pair mechanism (RPM).

NIN-like protein 7 transcription factor is a plant nitrate sensor.

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants' primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.

Plant vernalization proteins contain unusual PHD superdomains without histone H3 binding activity.

PHD fingers are modular domains in chromatin-associated proteins that decode the methylation status of histone H3 tails. A PHD finger signature is found in plant vernalization (VEL) proteins, which function as accessory factors of the Polycomb system to control flowering in Arabidopsis through an epigenetic silencing mechanism. It has been proposed that VEL PHD fingers bind to methylated histone H3 tails to facilitate association of the Polycomb silencing machinery with target genes. Here, we use structural analysis by X-ray crystallography to show that the VEL PHD finger forms the central module of a larger compact tripartite superdomain that also contains a zinc finger and a four-helix bundle. This PHD superdomain fold is only found in one other family, the OBERON proteins, which have multiple functions in Arabidopsis meristems to control plant growth. The putative histone-binding surface of OBERON proteins exhibits the characteristic three-pronged pocket of histone-binding PHD fingers and binds to methylated histone H3 tails. However, that of VEL PHD fingers lacks this architecture and exhibits unusually high positive surface charge. This VEL PHD superdomain neither binds to unmodified nor variously modified histone H3 tails, as demonstrated by isothermal calorimetry and NMR spectroscopy. Instead, the VEL PHD superdomain interacts with negatively charged polymers. Our evidence argues for evolution of a divergent function for the PHD superdomain in vernalization that does not involve histone tail decoding.

The TOR complex controls ATP levels to regulate actin cytoskeleton dynamics in Arabidopsis.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B, or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.

Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

MAIN CONCLUSION: Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.

Aerial (+)-borneol modulates root morphology, auxin signalling and meristematic activity in Arabidopsis roots.

One of the characteristic aspects of odour sensing in humans is the activation of olfactory receptors in a slightly different manner in response to different enantiomers. Here, we focused on whether plants showed enantiomer-specific response similar to that in humans. We exposed Arabidopsis seedlings to methanol (control) and (+)- or (-)-borneol, and found that only (+)-borneol reduced the root length. Furthermore, the root-tip width was more increased upon (+)-borneol exposure than upon (-)-borneol exposure. In addition, root-hair formation was observed near the root tip in response to (+)-borneol. Auxin signalling was strongly reduced in the root tip following exposure to (+)-borneol, but was detected following exposure to (-)-borneol and methanol. Similarly, in the root tip, the activity of cyclin B1:1 was detected on exposure to (-)-borneol and methanol, but not on exposure to (+)-borneol, indicating that (+)-borneol inhibits the meristematic activity in the root. These results partially explain the (+)-borneol-specific reduction in the root length of Arabidopsis. Our results indicate the presence of a sensing system specific for (+)-borneol in Arabidopsis.

Potent inhibition of TCP transcription factors by miR319 ensures proper root growth in Arabidopsis.

KEY MESSAGE: Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.

KARRIKIN INSENSITIVE2 regulates leaf development, root system architecture and arbuscular-mycorrhizal symbiosis in Brachypodium distachyon.

KARRIKIN INSENSITIVE2 (KAI2) is an α/ß-hydrolase required for plant responses to karrikins, which are abiotic butenolides that can influence seed germination and seedling growth. Although represented by four angiosperm species, loss-of-function kai2 mutants are phenotypically inconsistent and incompletely characterised, resulting in uncertainties about the core functions of KAI2 in plant development. Here we characterised the developmental functions of KAI2 in the grass Brachypodium distachyon using molecular, physiological and biochemical approaches. Bdkai2 mutants exhibit increased internode elongation and reduced leaf chlorophyll levels, but only a modest increase in water loss from detached leaves. Bdkai2 shows increased numbers of lateral roots and reduced root hair growth, and fails to support normal root colonisation by arbuscular-mycorrhizal (AM) fungi. The karrikins KAR1 and KAR2 , and the strigolactone (SL) analogue rac-GR24, each elicit overlapping but distinct changes to the shoot transcriptome via BdKAI2. Finally, we show that BdKAI2 exhibits a clear ligand preference for desmethyl butenolides and weak responses to methyl-substituted SL analogues such as GR24. Our findings suggest that KAI2 has multiple roles in shoot development, root system development and transcriptional regulation in grasses. Although KAI2-dependent AM symbiosis is likely conserved within monocots, the magnitude of the effect of KAI2 on water relations may vary across angiosperms.

Towards resolution of a paradox in plant G-protein signaling.

G-proteins are molecular on-off switches that are involved in transmitting a variety of extracellular signals to their intracellular targets. In animal and yeast systems, the switch property is encoded through nucleotides: a GDP-bound state is the "off-state" and the GTP-bound state is the "on-state". The G-protein cycle consists of the switch turning on through nucleotide exchange facilitated by a G-protein coupled receptor and the switch turning off through hydrolysis of GTP back to GDP, facilitated by a protein designated REGULATOR OF G SIGNALING 1 (RGS). In plants, G-protein signaling dramatically differs from that in animals and yeast. Despite stringent conservation of the nucleotide binding and catalytic structures over the 1.6 billion years that separate the evolution of plants and animals, genetic and biochemical data indicate that nucleotide exchange is less critical for this switch to operate in plants. Also, the loss of the single RGS protein in Arabidopsis (Arabidopsis thaliana) confers unexpectedly weaker phenotypes consistent with a diminished role for the G cycle, at least under static conditions. However, under dynamic conditions, genetic ablation of RGS in Arabidopsis results in a strong phenotype. We explore explanations to this conundrum by formulating a mathematical model that takes into account the accruing evidence for the indispensable role of phosphorylation in G-protein signaling in plants and that the G-protein cycle is needed to process dynamic signal inputs. We speculate that the plant G-protein cycle and its attendant components evolved to process dynamic signals through signaling modulation rather than through on-off, switch-like regulation of signaling. This so-called change detection may impart greater fitness for plants due to their sessility in a dynamic light, temperature, and pest environment.

Early sensing of phosphate deprivation triggers the formation of extra root cap cell layers via SOMBRERO through a process antagonized by auxin signaling.

KEY MESSAGE: The role of the root cap in the plant response to phosphate deprivation has been scarcely investigated. Here we describe early structural, physiological and molecular changes prior to the determinate growth program of the primary roots under low Pi and unveil a critical function of the transcription factor SOMBRERO in low Pi sensing. Mineral nutrient distribution in the soil is uneven and roots efficiently adapt to improve uptake and assimilation of sparingly available resources. Phosphate (Pi) accumulates in the upper layers and thus short and branched root systems proliferate to better exploit organic and inorganic Pi patches. Here we report an early adaptive response of the Arabidopsis primary root that precedes the entrance of the meristem into the determinate developmental program that is a hallmark of the low Pi sensing mechanism. In wild-type seedlings transferred to low Pi medium, the quiescent center domain in primary root tips increases as an early response, as revealed by WOX5:GFP expression and this correlates with a thicker root tip with extra root cap cell layers. The halted primary root growth in WT seedlings could be reversed upon transfer to medium supplemented with 250 µM Pi. Mutant and gene expression analysis indicates that auxin signaling negatively affects the cellular re-specification at the root tip and enabled identification of the transcription factor SOMBRERO as a critical element that orchestrates both the formation of extra root cap layers and primary root growth under Pi scarcity. Moreover, we provide evidence that low Pi-induced root thickening or the loss-of-function of SOMBRERO is associated with expression of phosphate transporters at the root tip. Our data uncover a developmental window where the root tip senses deprivation of a critical macronutrient to improve adaptation and surveillance.

Spatial differences in stoichiometry of EGR phosphatase and Microtubule-associated Stress Protein 1 control root meristem activity during drought stress.

During moderate severity drought and low water potential (ψw) stress, poorly understood signaling mechanisms restrict both meristem cell division and subsequent cell expansion. We found that the Arabidopsis thaliana Clade E Growth-Regulating 2 (EGR2) protein phosphatase and Microtubule-Associated Stress Protein 1 (MASP1) differed in their stoichiometry of protein accumulation across the root meristem and had opposing effects on root meristem activity at low ψw. Ectopic MASP1 or EGR expression increased or decreased, respectively, root meristem size and root elongation during low ψw stress. This, along with the ability of phosphomimic MASP1 to overcome the EGR-mediated suppression of root meristem size and the observation that ectopic EGR expression had no effect on unstressed plants, indicated that during low ψw EGR activation and attenuation of MASP1 phosphorylation in their overlapping zone of expression determines root meristem size and activity. Ectopic EGR expression also decreased root cell size at low ψw. Conversely, both the egr1-1 egr2-1 and egr1-1 egr2-1 masp1-1 mutants had similarly increased root cell size but only egr1-1egr2-1 had increased cell division. These observations demonstrated that EGRs affect meristem activity via MASP1 but affect cell expansion via other mechanisms. Interestingly, EGR2 was highly expressed in the root cortex, a cell type important for growth regulation and environmental response.

Redox-mediated structural and functional switching of C-repeat binding factors enhances plant cold tolerance.

C-repeat binding factors (CBFs) are key cold-responsive transcription factors that play pleiotropic roles in the cold acclimation, growth, and development of plants. Cold-sensitive cbf knockout mutants and cold-tolerant CBF overexpression lines exhibit abnormal phenotypes at warm temperatures, suggesting that CBF activity is precisely regulated, and a critical threshold level must be maintained for proper plant growth under normal conditions. Cold-inducible CBFs also exist in warm-climate plants but as inactive disulfide-bonded oligomers. However, upon translocation to the nucleus under a cold snap, the h2-isotype of cytosolic thioredoxin (Trx-h2), reduces the oxidized (inactive) CBF oligomers and the newly synthesized CBF monomers, thus producing reduced (active) CBF monomers. Thus, the redox-dependent structural switching and functional activation of CBFs protect plants under cold stress.