Circadian clocks coordinate physiology and development as an adaption to the oscillating day/night cycle caused by the rotation of Earth on its axis and the changing length of day and night away from the equator caused by orbiting the sun. Circadian clocks confer advantages by entraining to rhythmic environmental cycles to ensure that internal events within the plant occur at the correct time with respect to the cyclic external environment. Advances in determining the structure of circadian oscillators and the pathways that allow them to respond to light, temperature, and metabolic signals have begun to provide a mechanistic insight to the process of entrainment in Arabidopsis (Arabidopsis thaliana). We describe the concepts of entrainment and how it occurs. It is likely that a thorough mechanistic understanding of the genetic and physiological basis of circadian entrainment will provide opportunities for crop improvement.

Like many organisms, plants have evolved a genetic network, the circadian clock, to coordinate processes with day/night cycles. In plants, the clock is a pervasive regulator of development and modulates many aspects of physiology. Clock-regulated processes range from the correct timing of growth and cell division to interactions with the root microbiome. Recently developed techniques, such as single-cell time-lapse microscopy and single-cell RNA-seq, are beginning to revolutionize our understanding of this clock regulation, revealing a surprising degree of organ, tissue, and cell-type specificity. In this review, we highlight recent advances in our spatial view of the clock across the plant, both in terms of how it is regulated and how it regulates a diversity of output processes. We outline how understanding these spatially specific functions will help reveal the range of ways that the clock provides a fitness benefit for the plant.

The plant circadian clock coordinates developmental, physiological, and metabolic processes with diel changes in light and temperature throughout the year. The balance between the persistence and plasticity of the clock in response to predictable and unpredictable environmental changes may be key to the clock's adaptive nature across temporal and spatial scales. Studies under controlled conditions have uncovered critical signaling pathways involved in light and temperature perception by the clock; however, they don't account for the natural lag of temperature behind photoperiod. Studies in natural environments provide key insights into the clock's adaptive advantage under more complex natural settings. Here, we discuss the role of the circadian clock in light and temperature perception and signaling, how the clock integrates these signals for a coordinated and adaptive response, and the adaptive advantage conferred by the clock across time and space in natural environments.

Circadian clocks govern temporal programs in the green lineage (Chloroplastida) as they do in other photosynthetic pro- and eukaryotes, bacteria, fungi, animals, and humans. Their physiological properties, including entrainment, phase responses, and temperature compensation, are well conserved. The involvement of transcriptional/translational feedback loops in the oscillatory machinery and reversible phosphorylation events are also maintained. Circadian clocks control a large variety of output rhythms in green algae and terrestrial plants, adjusting their metabolism and behavior to the day-night cycle. The angiosperm Arabidopsis (Arabidopsis thaliana) represents a well-studied circadian clock model. Several molecular components of its oscillatory machinery are conserved in other Chloroplastida, but their functions may differ. Conserved clock components include at least one member of the CIRCADIAN CLOCK ASSOCIATED1/REVEILLE and one of the PSEUDO RESPONSE REGULATOR family. The Arabidopsis evening complex members EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHMO are found in the moss Physcomitrium patens and in the liverwort Marchantia polymorpha. In the flagellate chlorophyte alga Chlamydomonas reinhardtii, only homologs of ELF4 and LUX (named RHYTHM OF CHLOROPLAST ROC75) are present. Temporal ROC75 expression in C. reinhardtii is opposite to that of the angiosperm LUX, suggesting different clock mechanisms. In the picoalga Ostreococcus tauri, both ELF genes are missing, suggesting that it has a progenitor circadian "green" clock. Clock-relevant photoreceptors and thermosensors vary within the green lineage, except for the CRYPTOCHROMEs, whose variety and functions may differ. More genetically tractable models of Chloroplastida are needed to draw final conclusions about the gradual evolution of circadian clocks within the green lineage.

Vernalization, influenced by environmental factors, is an essential process associated with the productivity of temperate crops, during which epigenetic regulation of gene expression plays an important role. Although DNA methylation is one of the major epigenetic mechanisms associated with the control of gene expression, global changes in DNA methylation in the regulation of gene expression during vernalization-induced flowering of temperate plants remain largely undetermined. To characterize vernalization-associated DNA methylation dynamics, we performed whole-genome bisulfite-treated sequencing and transcriptome sequencing in orchardgrass (Dactylis glomerata) during vernalization. The results revealed that increased levels of genome DNA methylation during the early vernalization of orchardgrass were associated with transcriptional changes in DNA methyltransferase and demethylase genes. Upregulated expression of vernalization-related genes during early vernalization was attributable to an increase in mCHH in the promoter regions of these genes. Application of an exogenous DNA methylation accelerator or overexpression of orchardgrass NUCLEAR POLY(A) POLYMERASE (DgPAPS4) promoted earlier flowering, indicating that DNA hypermethylation plays an important role in vernalization-induced flowering. Collectively, our findings revealed that vernalization-induced hypermethylation is responsible for floral primordium initiation and development. These observations provide a theoretical foundation for further studies on the molecular mechanisms underlying the control of vernalization in temperate grasses.

Comment in doi: 10.1093/plphys/kiac348.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

Grain size is one of the crucial factors determining grain yield. However, the genetic and molecular mechanisms of florigen repression complexes (FRCs) underlying grain size in rice (Oryza sativa L.) have not been reported. Here, we report that the rice CENTRORADIALIS (CEN) family member OsCEN2 (also known as Rice TFL1/CEN homolog, RCN1), a phosphatidylethanolamine-binding protein (PEBP) family protein, negatively controls grain size in rice. Overexpression of OsCEN2 led to small grains, and knockout of OsCEN2 resulted in large, heavy grains. OsCEN2 influenced grain size by restricting cell expansion in the spikelet hull and seed filling. In in vivo and in vitro experiments, OsCEN2 physically interacted with a G-box factor 14-3-3 homolog, GF14f, which negatively regulates grain size. Bimolecular fluorescence complementation and yeast two-hybrid assays revealed that GF14f directly interacts with the basic leucine zipper (bZIP) transcription factor, OsFD2. Plants overexpressing OsFD2 produced smaller and lighter grains than wild-type plants. We found that OsFD2 also influences grain size by controlling cell expansion and division in the spikelet hull. Our results reveal the molecular mechanisms of the OsCEN2-GF14f-OsFD2 regulatory module in controlling grain size. Additionally, our study provides insight into the functions of the FRC in rice and suggests a strategy for improving seed size and weight.

Salinity and soil drying are expected to induce salt accumulation at the root-soil interface of transpiring plants. However, the consequences of this on the relationship between transpiration rate (E) and leaf xylem water potential (ψleaf-x) are yet to be quantified. Here, we used a noninvasive root pressure chamber to measure the E(ψleaf-x) relationship of tomato (Solanum lycopersicum L.) treated with (saline) or without 100-mM NaCl (nonsaline conditions). The results were reproduced and interpreted with a soil-plant hydraulic model. Under nonsaline conditions, the E(ψleaf-x) relationship became progressively more nonlinear as the soil dried (θ ≤ 0.13 cm3 cm-3, ψsoil = -0.08 MPa or less). Under saline conditions, plants exhibited an earlier nonlinearity in the E(ψleaf-x) relationship (θ ≤ 0.15 cm3 cm-3, ψsoil =  -0.05 MPa or less). During soil drying, salinity induced a more negative ψleaf-x at predawn, reduced transpiration rate, and caused a reduction in root hydraulic conductance (from 1.48 × 10-6 to 1.30 × 10-6 cm3 s-1 hPa-1). The model suggested that the marked nonlinearity was caused by salt accumulation at the root surface and the consequential osmotic gradients. In dry soil, most water potential dissipation occurred in the bulk soil and rhizosphere rather than inside the plant. Under saline-dry conditions, the loss in osmotic potential at the root surface was the preeminent component of the total dissipation. The physical model of water flow and solute transport supports the hypothesis that a buildup of osmotic potential at the root-soil interface causes a large drop in ψleaf-x and limits transpiration rate under drought and salinity.

Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE (RVE) proteins are recently discovered members of the plant circadian circuitry that activate the evening complex and PSEUDO-RESPONSE REGULATOR genes to maintain regular circadian oscillation. The RVE8 protein and its two homologs, RVE 4 and 6 in Arabidopsis (Arabidopsis thaliana), have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size, and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw new connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid, and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset provides substantial insight into the far-reaching impact RVE8-like proteins have on the diel plant cell environment.

Serine protease subtilase, found widely in both eukaryotes and prokaryotes, participates in various biological processes. However, how fungal subtilase regulates plant immunity is a major concern. Here, we identified a secreted fungal subtilase, UvPr1a, from the rice false smut (RFS) fungus Ustilaginoidea virens. We characterized UvPr1a as a virulence effector localized to the plant cytoplasm that inhibits plant cell death induced by Bax. Heterologous expression of UvPr1a in rice (Oryza sativa) enhanced plant susceptibility to rice pathogens. UvPr1a interacted with the important rice protein SUPPRESSOR OF G2 ALLELE OF skp1 (OsSGT1), a positive regulator of innate immunity against multiple rice pathogens, degrading OsSGT1 in a protease activity-dependent manner. Furthermore, host-induced gene silencing of UvPr1a compromised disease resistance of rice plants. Our work reveals a previously uncharacterized fungal virulence strategy in which a fungal pathogen secretes a subtilase to interfere with rice immunity through degradation of OsSGT1, thereby promoting infection. These genetic resources provide tools for introducing RFS resistance and further our understanding of plant-pathogen interactions.

Cell wall lignification is a key step in forming functional endodermis and protoxylem (PX) in plant roots. Lignified casparian strips (CS) in endodermis and tracheary elements of PX are essential for selective absorption and transport of water and nutrients. Although multiple key regulators of CS and PX have been identified, the spatial information that drives the developmental shift to root lignification remains unknown. Here, we found that brassinosteroid (BR) signaling plays a key role in inhibiting root lignification in the root elongation zone. The inhibitory activity of BR signaling occurs partially through the direct binding of BRASSINAZOLE-RESISTANT 1 (BZR1) to SHORT-ROOT (SHR), repressing the SHR-mediated activation of downstream genes that are involved in root lignification. Upon entering the mature root zone, BR signaling declines rapidly, which releases SHR activity and initiates root lignification. Our results provide a mechanistic view of the developmental transition to cell wall lignification in Arabidopsis thaliana roots.

Erratum in Plant Physiol. 2022 Oct 07;:

Light is essential to plant survival and elicits a wide range of plant developmental and physiological responses under different light conditions. A low red-to-far red (R/FR) light ratio induces shade-avoidance responses, including decreased anthocyanin accumulation, whereas a high R/FR light ratio promotes anthocyanin biosynthesis. However, the detailed molecular mechanism underpinning how different R/FR light ratios regulate anthocyanin homeostasis remains elusive, especially in non-model species. Here, we demonstrate that a low R/FR light ratio induced the expression of CmMYB4, which suppressed the anthocyanin activator complex CmMYB6-CmbHLH2, leading to the reduction of anthocyanin accumulation in Chrysanthemum (Chrysanthemum morifolium) petals. Specifically, CmMYB4 recruited the corepressor CmTPL (TOPLESS) to directly bind the CmbHLH2 promoter and suppressed its transcription by impairing histone H3 acetylation. Moreover, the low R/FR light ratio inhibited the PHYTOCHROME INTERACTING FACTOR family transcription factor CmbHLH16, which can competitively bind to CmMYB4 and destabilize the CmMYB4-CmTPL protein complex. Under the high R/FR light ratio, CmbHLH16 was upregulated, which impeded the formation of the CmMYB4-CmTPL complex and released the suppression of CmbHLH2, thus promoting anthocyanin accumulation in Chrysanthemum petals. Our findings reveal a mechanism by which different R/FR light ratios fine-tune anthocyanin homeostasis in flower petals.

Comment in doi: 10.1093/plphys/kiac347.

Ammonium uptake at plant roots is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Phosphorylation by the protein kinase calcineurin B-like protein (CBL)-interacting protein kinase 23 (CIPK23) transiently inactivates ammonium transporters (AMT1s), but the phosphatases activating AMT1s remain unknown. Here, we identified the PP2C phosphatase abscisic acid (ABA) insensitive 1 (ABI1) as an activator of AMT1s in Arabidopsis (Arabidopsis thaliana). We showed that high external ammonium concentrations elevate the level of the stress phytohormone ABA, possibly by de-glycosylation. Active ABA was sensed by ABI1-PYR1-like () complexes followed by the inactivation of ABI1, in turn activating CIPK23. Under favorable growth conditions, ABI1 reduced AMT1;1 and AMT1;2 phosphorylation, both by binding and inactivating CIPK23. ABI1 further directly interacted with AMT1;1 and AMT1;2, which would be a prerequisite for dephosphorylation of the transporter by ABI1. Thus, ABI1 is a positive regulator of ammonium uptake, coupling nutrient acquisition to abiotic stress signaling. Elevated ABA reduces ammonium uptake during stress situations, such as ammonium toxicity, whereas ABI1 reactivates AMT1s under favorable growth conditions.

The identification of drought stress regulatory genes is crucial for the genetic improvement of maize (Zea mays L.) yield. Nuclear factors Y (NF-Ys) are important transcription factors, but their roles in the drought stress tolerance of plants and underlying molecular mechanisms are largely unknown. In this work, we used yeast two-hybrid screening to identify potential interactors of ZmNF-YB16 and confirmed the interaction between ZmNF-YA1 and ZmNF-YB16-YC17 and between ZmNF-YA7 and ZmNF-YB16-YC17. ZmNF-YB16 interacted with ZmNF-YC17 via its histone fold domain to form a heterodimer in the cytoplasm and then entered the nucleus to form a heterotrimer with ZmNF-YA1 or ZmNF-YA7 under osmotic stress. Overexpression of ZmNF-YA1 improved drought and salt stress tolerance and root development of maize, whereas zmnf-ya1 mutants exhibited drought and salt stress sensitivity. ZmNF-YA1-mediated transcriptional regulation, especially in JA signaling, histone modification, and chromatin remodeling, could underlie the altered stress tolerance of zmnf-ya1 mutant plants. ZmNF-YA1 bound to promoter CCAAT motifs and directly regulated the expression of multiple genes that play important roles in stress responses and plant development. Comparison of ZmNF-YB16- and ZmNF-YA1-regulated genes showed that ZmNF-YA1 and ZmNF-YB16 have similar biological functions in stress responses but varied functions in other biological processes. Taken together, ZmNF-YA1 is a positive regulator of plant drought and salt stress responses and is involved in the root development of maize, and ZmNF-Y complexes with different subunits may have discrepant functions.

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater, and intertidal environments. The latter can experience substantial long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We found that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations corresponded with the rate of temperature decrease. We did not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance but showed that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As intertidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signaling interacts with osmotic signaling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signaling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca2+/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.

Circadian clocks facilitate the coordination of physiological and developmental processes to changing daily and seasonal cycles. A hub for environmental signaling pathways in the Arabidopsis (Arabidopsis thaliana) circadian clock is the evening complex (EC), a protein complex composed of EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRYTHMO (LUX). Formation of the EC depends on ELF3, a scaffold protein that recruits the other components of the EC and chromatin remodeling enzymes to repress gene expression. Regulating the cellular distribution of ELF3 is thus an important mechanism in controlling its activity. Here, we determined that the cellular and sub-nuclear localization of ELF3 is responsive to red (RL) and blue light and that these two wavelengths have apparently competitive effects on where in the cell ELF3 localizes. We further characterized the RL response, revealing that at least two RL pathways influence the cellular localization of ELF3. One of these depends on the RL photoreceptor phytochrome B (phyB), while the second is at least partially independent of phyB activity. Finally, we investigated how changes in the cellular localization of ELF3 are associated with repression of EC target-gene expression. Our analyses revealed a complex effect whereby ELF3 is required for controlling RL sensitivity of morning-phased genes, but not evening-phased genes. Together, our findings establish a previously unknown mechanism through which light signaling influences ELF3 activity.

Magnesium (Mg) is an essential metal for chlorophyll biosynthesis and other metabolic processes in plant cells. Mg is largely stored in the vacuole of various cell types and remobilized to meet cytoplasmic demand. However, the transport proteins responsible for mobilizing vacuolar Mg2+ remain unknown. Here, we identified two Arabidopsis (Arabidopsis thaliana) Mg2+ transporters (MAGNESIUM TRANSPORTER 1 and 2; MGT1 and MGT2) that facilitate Mg2+ mobilization from the vacuole, especially when external Mg supply is limited. In addition to a high degree of sequence similarity, MGT1 and MGT2 exhibited overlapping expression patterns in Arabidopsis tissues, implying functional redundancy. Indeed, the mgt1 mgt2 double mutant, but not mgt1 and mgt2 single mutants, showed exaggerated growth defects as compared to the wild type under low-Mg conditions, in accord with higher expression levels of Mg-starvation gene markers in the double mutant. However, overall Mg level was also higher in mgt1 mgt2, suggesting a defect in Mg2+ remobilization in response to Mg deficiency. Consistently, MGT1 and MGT2 localized to the tonoplast and rescued the yeast (Saccharomyces cerevisiae) mnr2Δ (manganese resistance 2) mutant strain lacking the vacuolar Mg2+ efflux transporter. In addition, disruption of MGT1 and MGT2 suppressed high-Mg sensitivity of calcineurin B-like 2 and 3 (cbl2 cbl3), a mutant defective in vacuolar Mg2+ sequestration, suggesting that vacuolar Mg2+ influx and efflux processes are antagonistic in a physiological context. We further crossed mgt1 mgt2 with mgt6, which lacks a plasma membrane MGT member involved in Mg2+ uptake, and found that the triple mutant was more sensitive to low-Mg conditions than either mgt1 mgt2 or mgt6. Hence, Mg2+ uptake (via MGT6) and vacuolar remobilization (through MGT1 and MGT2) work synergistically to achieve Mg2+ homeostasis in plants, especially under low-Mg supply in the environment.

Calmodulin-like proteins (CMLs) are calcium (Ca2+) sensors involved in plant growth and development as well as adaptation to environmental stresses; however, their roles in plant responses to cold are not well understood. To reveal the role of MsCML10 from alfalfa (Medicago sativa) in regulating cold tolerance, we examined transgenic alfalfa and Medicago truncatula overexpressing MsCML10, MsCML10-RNAi alfalfa, and a M. truncatula cml10-1 mutant and identified MsCML10-interacting proteins. MsCML10 and MtCML10 transcripts were induced by cold treatment. Upregulation or downregulation of MsCML10 resulted in increased or decreased cold tolerance, respectively, while cml10-1 showed decreased cold tolerance that was complemented by expressing MsCML10, suggesting that MsCML10 regulates cold tolerance. MsCML10 interacted with glutathione S-transferase (MsGSTU8) and fructose 1,6-biphosphate aldolase (MsFBA6), and the interaction depended on the presence of Ca2+. The altered activities of Glutathione S-transferase and FBA and levels of ROS and sugars were associated with MsCML10 transcript levels. We propose that MsCML10 decodes the cold-induced Ca2+ signal and regulates cold tolerance through activating MsGSTU8 and MsFBA6, leading to improved maintenance of ROS homeostasis and increased accumulation of sugars for osmoregulation, respectively.

Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lettuce (Lactuca sativa) cv. Grand Rapids germinates in response to nanomolar karrikinolide (KAR1). Lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), which in Arabidopsis (Arabidopsis thaliana) encodes a receptor that is required for KAR responses. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of imbibition. Through cross-species complementation assays in Arabidopsis, we found that an LsKAI2b transgene confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of KAI2 proteins from lettuce and a fire-follower, whispering bells (Emmenanthe penduliflora). This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand specificity of KAI2. Further support for the importance of these residues was found through a broader comparison of pocket residues among 281 KAI2 proteins from 184 asterid species. Almost all KAI2 proteins had either Tyr or Phe identity at position 124. Genes encoding Y124-type KAI2 are more broadly distributed in asterids than in F124-type KAI2. Substitutions at residues 96, 124, 139, and 161 in Arabidopsis KAI2 produced a broad array of responses to KAR1, KAR2, and rac-GR24. This suggests that the diverse ligand preferences observed among KAI2 proteins in plants could have evolved through relatively few mutations.

Comment in doi: 10.1093/plphys/kiac318.

The circadian clock facilitates the survival and reproduction of crop plants under harsh environmental conditions such as drought and osmotic and salinity stresses, mainly by reprogramming the endogenous transcriptional landscape. Nevertheless, the genome-wide roles of core clock components in rice (Oryza sativa L.) abiotic stress tolerance are largely uncharacterized. Here, we report that CIRCADIAN CLOCK ASSOCIATED1 (OsCCA1), a vital clock component in rice, is required for tolerance to salinity, osmotic, and drought stresses. DNA affinity purification sequencing coupled with transcriptome analysis identified 692 direct transcriptional target genes of OsCCA1. Among them, the genes involved in abscisic acid (ABA) signaling, including group A protein phosphatase 2C genes and basic region and leucine zipper 46 (OsbZIP46), were substantially enriched. Moreover, OsCCA1 could directly bind the promoters of OsPP108 and OsbZIP46 to activate their expression. Consistently, oscca1 null mutants generated via genome editing displayed enhanced sensitivities to ABA signaling. Together, our findings illustrate that OsCCA1 confers multiple abiotic stress tolerance likely by orchestrating ABA signaling, which links the circadian clock with ABA signaling in rice.

In C4 plants, the pyruvate (Pyr), phosphate dikinase regulatory protein (PDRP) regulates the activity of the C4 pathway enzyme Pyr, phosphate dikinase (PPDK) in a light-/dark-dependent manner. The importance of this regulatory action to C4 pathway function and overall C4 photosynthesis is unknown. To resolve this question, we assessed in vivo PPDK phospho-regulation and whole leaf photophysiology in a CRISPR-Cas9 PDRP knockout (KO) mutant of the NADP-ME C4 grass green millet (Setaria viridis). PDRP enzyme activity was undetectable in leaf extracts from PDRP KO lines. Likewise, PPDK phosphorylated at the PDRP-regulatory Thr residue was immunologically undetectable in leaf extracts. PPDK enzyme activity in rapid leaf extracts was constitutively high in the PDRP KO lines, irrespective of light or dark pretreatment of leaves. Gas exchange analysis of net CO2 assimilation revealed PDRP KO leaves had markedly slower light induction kinetics when leaves transition from dark to high-light or low-light to high-light. In the initial 30 min of the light induction phase, KO leaves had an ∼15% lower net CO2 assimilation rate versus the wild-type (WT). Despite the impaired slower induction kinetics, we found growth and vigor of the KO lines to be visibly indistinguishable from the WT when grown in normal air and under standard growth chamber conditions. However, the PDRP KO plants grown under a fluctuating light regime exhibited a gradual multi-day decline in Fv/Fm, indicative of progressive photosystem II damage due to the absence of PDRP. Collectively, our results demonstrate that one of PDRP's functions in C4 photosynthesis is to ensure optimal photosynthetic light induction kinetics during dynamic changes in incident light.

Comment in doi: 10.1093/plphys/kiac349.

The circadian clock helps organisms to anticipate and coordinate gene regulatory responses to changes in environmental stimuli. Under stresses, both time of day and the circadian clock closely control the magnitude of plant responses. The identification of clock-regulated genes is, therefore, important when studying the influence of environmental factors. Here, we present CAST-R (Circadian And heat STress-Responsive), a "Shiny" application that allows users to identify and visualize circadian and heat stress-responsive genes in plants. More specifically, users can generate and export profiles and heatmaps representing transcript abundance of a single or of multiple Arabidopsis (Arabidopsis thaliana) genes over a 24-h time course, in response to heat stress and during recovery following the stress. The application also takes advantage of published Arabidopsis chromatin immunoprecipitation-sequencing datasets to visualize the connections between clock proteins and their targets in an interactive network. In addition, CAST-R offers the possibility to perform phase (i.e. timing of expression) enrichment analyses for rhythmic datasets from any species, within and beyond plants. This functionality combines statistical analyses and graphical representations to identify significantly over- and underrepresented phases within a subset of genes. Lastly, profiles of transcript abundance can be visualized from multiple circadian datasets generated in Arabidopsis, Brassica rapa, barley (Hordeum vulgare), and rice (Oryza sativa). In summary, CAST-R is a user-friendly interface that allows the rapid identification of circadian and stress-responsive genes through multiple modules of visualization. We anticipate that this tool will make it easier for users to obtain temporal and dynamic information on genes of interest that links plant responses to environmental signals.

The circadian clock is conserved at both the level of transcriptional networks as well as core genes in plants, ensuring that biological processes are phased to the correct time of day. In the model plant Arabidopsis (Arabidopsis thaliana), the core circadian SHAQKYF-type-MYB (sMYB) genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and REVEILLE (RVE4) show genetic linkage with PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7, respectively. Leveraging chromosome-resolved plant genomes and syntenic ortholog analysis enabled tracing this genetic linkage back to Amborella trichopoda, a sister lineage to the angiosperm, and identifying an additional evolutionarily conserved genetic linkage in light signaling genes. The LHY/CCA1-PRR5/9, RVE4/8-PRR3/7, and PIF3-PHYA genetic linkages emerged in the bryophyte lineage and progressively moved within several genes of each other across an array of angiosperm families representing distinct whole-genome duplication and fractionation events. Soybean (Glycine max) maintained all but two genetic linkages, and expression analysis revealed the PIF3-PHYA linkage overlapping with the E4 maturity group locus was the only pair to robustly cycle with an evening phase, in contrast to the sMYB-PRR morning and midday phase. While most monocots maintain the genetic linkages, they have been lost in the economically important grasses (Poaceae), such as maize (Zea mays), where the genes have been fractionated to separate chromosomes and presence/absence variation results in the segregation of PRR7 paralogs across heterotic groups. The environmental robustness model is put forward, suggesting that evolutionarily conserved genetic linkages ensure superior microhabitat pollinator synchrony, while wide-hybrids or unlinking the genes, as seen in the grasses, result in heterosis, adaptation, and colonization of new ecological niches.

Root exudates and rhizosheaths of attached soil are important features of growing roots. To elucidate factors involved in rhizosheath formation, wild-type (WT) barley (Hordeum vulgare L. cv. Pallas) and a root hairless mutant, bald root barley (brb), were investigated with a combination of physiological, biochemical, and immunochemical assays. When grown in soil, WT barley roots bound ∼5-fold more soil than brb per unit root length. High molecular weight (HMW) polysaccharide exudates of brb roots had less soil-binding capacity than those of WT root exudates. Carbohydrate and glycan monoclonal antibody analyses of HMW polysaccharide exudates indicated differing glycan profiles. Relative to WT plants, root exudates of brb had reduced signals for arabinogalactan-protein (AGP), extensin, and heteroxylan epitopes. In contrast, the root exudate of 2-week-old brb plants contained ∼25-fold more detectable xyloglucan epitope relative to WT. Root system immunoprints confirmed the higher levels of release of the xyloglucan epitope from brb root apices and root axes relative to WT. Epitope detection with anion-exchange chromatography indicated that the increased detection of xyloglucan in brb exudates was due to enhanced abundance of a neutral polymer. Conversely, brb root exudates contained decreased amounts of an acidic polymer, with soil-binding properties, containing the xyloglucan epitope and glycoprotein and heteroxylan epitopes relative to WT. We, therefore, propose that, in addition to physically structuring soil particles, root hairs facilitate rhizosheath formation by releasing a soil-binding polysaccharide complex.

ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.

Volatile esters are key compounds contributing to flavor intensity in commonly consumed fruits including apple (Malus domestica), strawberry (Fragaria spp.), and banana (Musa sapientum). In kiwifruit (Actinidia spp.), ethyl butanoate and other esters have been proposed to contribute fruity, sweet notes to commercial cultivars. Here, we investigated the genetic basis for ester production in Actinidia in an A. chinensis mapping population (AcMPO). A major quantitative trait loci for the production of multiple esters was identified at the high-flavor intensity (HiFI) locus on chromosome 20. This locus co-located with eight tandemly arrayed alcohol acyl transferase genes in the Red5 genome that were expressed in a ripening-specific fashion that corresponded with ester production. Biochemical characterization suggested two genes at the HiFI locus, alcohol acyl transferase 16-b/c (AT16-MPb/c), probably contributed most to the production of ethyl butanoate. A third gene, AT16-MPa, probably contributed more to hexyl butanoate and butyl hexanoate production, two esters that segregated in AcMPO. Sensory analysis of AcMPO indicated that fruit from segregating lines with high ester concentrations were more commonly described as being "fruity" as opposed to "beany". The downregulation of AT16-MPa-c by RNAi reduced ester production in ripe "Hort16A" fruit by >90%. Gas chromatography-olfactometry indicated the loss of the major "fruity" notes contributed by ethyl butanoate. A comparison of unimproved Actinidia germplasm with those of commercial cultivars indicated that the selection of fruit with high concentrations of alkyl esters (but not green note aldehydes) was probably an important selection trait in kiwifruit cultivation. Understanding ester production at the HiFI locus is a critical step toward maintaining and improving flavor intensity in kiwifruit.

Wheat stripe rust and powdery mildew are important worldwide diseases of wheat (Triticum aestivum). The wheat cultivar Xingmin318 (XM318) is resistant to both wheat stripe rust and powdery mildew, which are caused by Puccinia striiformis f. sp. tritici (Pst) and Blumeria graminis f. sp. tritici (Bgt), respectively. To explore the difference between wheat defense response against Pst and Bgt, quantitative proteomic analyses of XM318 inoculated with either Pst or Bgt were performed using tandem mass tags technology. A total of 741 proteins were identified as differentially accumulated proteins (DAPs). Bioinformatics analyses indicated that some functional categories, including antioxidant activity and immune system process, exhibited obvious differences between Pst and Bgt infections. Intriguingly, only 42 DAPs responded to both Pst and Bgt infections. Twelve DAPs were randomly selected for reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, and the mRNA expression levels of 11 were consistent with their protein expression. Furthermore, gene silencing using the virus-induced gene silencing system indicated that glutathione S-transferase (TaGSTU6) has an important role in resistance to Bgt but not to Pst. TaGSTU6 interacted with the cystathionine beta-synthase (CBS) domain-containing protein (TaCBSX3) in both Pst and Bgt infections. Knockdown of TaCBSX3 expression only reduced wheat resistance to Bgt infection. Overexpression of TaGSTU6 and TaCBSX3 in Arabidopsis (Arabidopsis thaliana) promoted plant resistance to Pseudomonas syringae pv. Tomato DC3000. Our results indicate that TaGSTU6 interaction with TaCBSX3 only confers wheat resistance to Bgt, suggesting that wheat has different response mechanisms to Pst and Bgt stress.

Basic helix-loop-helix (bHLH) transcription factors constitute a superfamily in eukaryotes, but their roles in plant immunity remain largely uncharacterized. We found that the transcript abundance in tomato (Solanum lycopersicum) leaves of one bHLH transcription factor-encoding gene, negative regulator of resistance to DC3000 1 (Nrd1), increased significantly after treatment with the immunity-inducing flgII-28 peptide. Plants carrying a loss-of-function mutation in Nrd1 (Δnrd1) showed enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 although early pattern-triggered immunity responses, such as generation of reactive oxygen species and activation of mitogen-activated protein kinases after treatment with flagellin-derived flg22 and flgII-28 peptides, were unaltered compared to wild-type plants. RNA-sequencing (RNA-seq) analysis identified a gene, Arabinogalactan protein 1 (Agp1), whose expression is strongly suppressed in an Nrd1-dependent manner. Agp1 encodes an arabinogalactan protein, and overexpression of the Agp1 gene in Nicotiana benthamiana led to ∼10-fold less Pst growth compared to the control. These results suggest that the Nrd1 protein promotes tomato susceptibility to Pst by suppressing the defense gene Agp1. RNA-seq also revealed that the loss of Nrd1 function has no effect on the transcript abundance of immunity-associated genes, including AvrPtoB tomato-interacting 9 (Bti9), Cold-shock protein receptor (Core), Flagellin sensing 2 (Fls2), Flagellin sensing (Fls3), and Wall-associated kinase 1 (Wak1) upon Pst inoculation, suggesting that the enhanced immunity observed in the Δnrd1 mutants is due to the activation of key PRR signaling components as well as the loss of Nrd1-regulated suppression of Agp1.

During and after the domestication of crops from ancestral wild plants, humans selected cultivars that could change their flowering time in response to seasonal daylength. Continuous selection of this trait eventually allowed the introduction of crops into higher or lower latitudes and different climates from the original regions where domestication initiated. In the past two decades, numerous studies have found the causal genes or alleles that change flowering time and have assisted in adapting crop species such as barley (Hordeum vulgare), wheat (Triticum aestivum L.), rice (Oryza sativa L.), pea (Pisum sativum L.), maize (Zea mays spp. mays), and soybean (Glycine max (L.) Merr.) to new environments. This updated review summarizes the genes or alleles that contributed to crop adaptation in different climatic areas. Many of these genes are putative orthologs of Arabidopsis (Arabidopsis thaliana) core clock genes. We also discuss how knowledge of the clock's molecular functioning can facilitate molecular breeding in the future.

Parasitism is a successful life strategy that has evolved independently in several families of vascular plants. The genera Cuscuta and Orobanche represent examples of the two profoundly different groups of parasites: one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs from Cuscuta campestris and Orobanche aegyptiaca. We showed that C. campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event. The exploratory analysis of the miRNA population (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic O. aegyptiaca also revealed a loss of a number of miRNAs compared to photosynthetic species from the same order. In summary, our study shows partly similar evolutionary signatures in the RNA silencing machinery in both parasites. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants.

The plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor family is most closely associated with regulating plant developmental programs. Recently, TCPs were also shown to mediate host immune signaling, both as targets of pathogen virulence factors and as regulators of plant defense genes. However, comprehensive characterization of TCP gene targets is still lacking. Loss of function of the class I TCP gene AtTCP8 attenuates early immune signaling and, when combined with mutations in AtTCP14 and AtTCP15, additional layers of defense signaling in Arabidopsis (Arabidopsis thaliana). Here, we focus on TCP8, the most poorly characterized of the three to date. We used chromatin immunoprecipitation and RNA sequencing to identify TCP8-bound gene promoters and differentially regulated genes in the tcp8 mutant; these datasets were heavily enriched in signaling components for multiple phytohormone pathways, including brassinosteroids (BRs), auxin, and jasmonic acid. Using BR signaling as a representative example, we showed that TCP8 directly binds and activates the promoters of the key BR transcriptional regulatory genes BRASSINAZOLE-RESISTANT1 (BZR1) and BRASSINAZOLE-RESISTANT2 (BZR2/BES1). Furthermore, tcp8 mutant seedlings exhibited altered BR-responsive growth patterns and complementary reductions in BZR2 transcript levels, while TCP8 protein demonstrated BR-responsive changes in subnuclear localization and transcriptional activity. We conclude that one explanation for the substantial targeting of TCP8 alongside other TCP family members by pathogen effectors may lie in its role as a modulator of BR and other plant hormone signaling pathways.