SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 Research Articles

PHOTOLYASE/BLUE LIGHT RECEPTOR2 regulates chrysanthemum flowering by compensating for gibberellin perception.

The gibberellins (GAs) receptor GA INSENSITIVE DWARF1 (GID1) plays a central role in GA signal perception and transduction. The typical photoperiodic plant chrysanthemum (Chrysanthemum morifolium) only flowers when grown in short-day photoperiods. In addition, chrysanthemum flowering is also controlled by the aging pathway, but whether and how GAs participate in photoperiod- and age-dependent regulation of flowering remain unknown. Here, we demonstrate that photoperiod affects CmGID1B expression in response to GAs and developmental age. Moreover, we identified PHOTOLYASE/BLUE LIGHT RECEPTOR2, an atypical photocleavage synthase, as a CRYPTOCHROME-INTERACTING bHLH1 interactor with which it forms a complex in response to short days to activate CmGID1B transcription. Knocking down CmGID1B raised endogenous bioactive GA contents and GA signal perception, in turn modulating the expression of the aging-related genes MicroRNA156 and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3. We propose that exposure to short days accelerates the juvenile-to-adult transition by increasing endogenous GA contents and response to GAs, leading to entry into floral transformation.

MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa.

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa's relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9-overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate.

MicroRNA156 conditions auxin sensitivity to enable growth plasticity in response to environmental changes in Arabidopsis

MicroRNAs (miRNAs) play diverse roles in plant development, but whether and how miRNAs participate in thermomorphogenesis remain ambiguous. Here we show that HYPONASTIC LEAVES 1 (HYL1)—a key component of miRNA biogenesis—acts downstream of the thermal regulator PHYTOCHROME INTERACTING FACTOR 4 in the temperature-dependent plasticity of hypocotyl growth in Arabidopsis. A hyl1-2 suppressor screen identified a dominant dicer-like1 allele that rescues hyl1-2’s defects in miRNA biogenesis and thermoresponsive hypocotyl elongation. Genome-wide miRNA and transcriptome analysis revealed microRNA156 (miR156) and its target SQUAMOSA PROMOTER-BINDING-PROTEIN-LIKE 9 (SPL9) to be critical regulators of thermomorphogenesis. Surprisingly, perturbation of the miR156/SPL9 module disengages seedling responsiveness to warm temperatures by impeding auxin sensitivity. Moreover, miR156-dependent auxin sensitivity also operates in the shade avoidance response at lower temperatures. Thus, these results unveil the miR156/SPL9 module as a previously uncharacterized genetic circuit that enables plant growth plasticity in response to environmental temperature and light changes.

Arabidopsis N6-methyladenosine methyltransferase FIONA1 regulates floral transition by affecting the splicing of FLC and the stability of floral activators SPL3 and SEP3.

N 6-methyladenosine (m6A) RNA methylation has been shown to play a crucial role in plant development and floral transition. Two recent studies have identified FIONA1 as an m6A methyltransferase that regulates the floral transition in Arabidopsis through influencing the stability of CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC). In this study, we confirmed that FIONA1 is an m6A methyltransferase that installs m6A marks in a small group of mRNAs. Furthermore, we show that, in addition to its role in influencing the stability of CO, SOC1, and FLC, FIONA1-mediated m6A methylation influences the splicing of FLC, a key floral repressor, and the stability of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3) and SEPALLATA3 (SEP3), floral activators, which together play a vital role in floral transition in Arabidopsis. Our study confirms the function of FIONA1 as an m6A methyltransferase and suggests a close molecular link between FIONA1-mediated m6A methylation and the splicing of FLC and the destabilization of SPL3 and SEP3 in flowering time control.

Two Arabidopsis MYB-SHAQKYF transcription repressors regulate leaf wax biosynthesis via transcriptional suppression on DEWAX.

Plant cuticular wax accumulation limits nonstomatal transpiration and is regulated by external environmental stresses. DEWAX (DECREASE WAX BIOSYNTHESIS) plays a vital role in diurnal wax biosynthesis. However, how DEWAX expression is controlled and the molecular mechanism of wax biosynthesis regulated by the diurnal cycle remains largely unknown. Here, we identified two Arabidopsis MYB-SHAQKYF transcription factors, MYS1 and MYS2, as new regulators in wax biosynthesis and drought tolerance. Mutations of both MYS1 and MYS2 caused significantly reduced leaf wax, whereas overexpression of MYS1 or MYS2 increased leaf wax biosynthesis and enhanced drought tolerance. Our results demonstrated that MYS1 and MYS2 act as transcription repressors and directly suppress DEWAX expression via ethylene response factor-associated amphiphilic repression motifs. Genetic interaction analysis with DEWAX, SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9), and CER1 (ECERIFERUM 1) in wax biosynthesis and under drought stresses demonstrated that MYS1 and MYS2 act upstream of the DEWAX-SPL9 module, thus regulating CER1 expression. Expression analysis suggested that the diurnal expression pattern of DEWAX is partly regulated by MYS1 and MYS2. Our findings demonstrate the roles of two unidentified transcription repressors, MYS1 and MYS2, in wax biosynthesis and provide insights into the mechanism of diurnal cycle-regulated wax biosynthesis.

Energy status-promoted growth and development of Arabidopsis require copper deficiency response transcriptional regulator SPL7.

Copper (Cu) is a cofactor of around 300 Arabidopsis proteins, including photosynthetic and mitochondrial electron transfer chain enzymes critical for adenosine triphosphate (ATP) production and carbon fixation. Plant acclimation to Cu deficiency requires the transcription factor SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7). We report that in the wild type (WT) and in the spl7-1 mutant, respiratory electron flux via Cu-dependent cytochrome c oxidase is unaffected under both normal and low-Cu cultivation conditions. Supplementing Cu-deficient medium with exogenous sugar stimulated growth of the WT, but not of spl7 mutants. Instead, these mutants accumulated carbohydrates, including the signaling sugar trehalose 6-phosphate, as well as ATP and NADH, even under normal Cu supply and without sugar supplementation. Delayed spl7-1 development was in agreement with its attenuated sugar responsiveness. Functional TARGET OF RAPAMYCIN and SNF1-RELATED KINASE1 signaling in spl7-1 argued against fundamental defects in these energy-signaling hubs. Sequencing of chromatin immunoprecipitates combined with transcriptome profiling identified direct targets of SPL7-mediated positive regulation, including Fe SUPEROXIDE DISMUTASE1 (FSD1), COPPER-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR1 (CITF1), and the uncharacterized bHLH23 (CITF2), as well as an enriched upstream GTACTRC motif. In summary, transducing energy availability into growth and reproductive development requires the function of SPL7. Our results could help increase crop yields, especially on Cu-deficient soils.

CITF1 Functions Downstream of SPL7 to Specifically Regulate Cu Uptake in Arabidopsis

Copper (Cu) is one of the most indispensable micronutrients, and proper Cu homeostasis is required for plants to maintain essential cellular functions. Plants activate the Cu uptake system during Cu limitation. Although SPL7 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7) and CITF1 (Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1) are two transcription factors in Cu homeostasis, it remains unclear how SPL7 and CITF1 control the Cu uptake system. Here, we reveal that overexpression of CITF1 causes the enhanced tolerance to Cu deficiency and the elevated expression of Cu uptake genes COPT2, FRO4 and FRO5. Electrophoretic mobility shift assays (EMSA) and transient expression assays indicate that SPL7 directly binds to and activates the promoter of CITF1. The overexpression of CITF1 partially rescues the sensitivity of spl7-1 to Cu deficiency. Transcriptome data suggest that SPL7 and CITF1 coregulate the Cu-homeostasis-signaling network, and CITF1 has its own independent functions. Moreover, both SPL7 and CITF1 can directly bind to and activate the promoters of three Cu uptake genes COPT2, FRO4 and FRO5. This work shows the functions of CITF1 in the Cu-homeostasis-signaling network, providing insights into the complicated molecular mechanism underlying Cu homeostasis.

Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases

Exploding seed pods evolved in the Arabidopsis relative Cardamine hirsuta via morphomechanical innovations that allow the storage and rapid release of elastic energy. Asymmetric lignin deposition within endocarpb cell walls is one such innovation that is required for explosive seed dispersal and evolved in association with the trait. However, the genetic control of this novel lignin pattern is unknown. Here, we identify three lignin-polymerizing laccases, LAC4, 11, and 17, that precisely colocalize with, and are redundantly required for, asymmetric lignification of endocarpb cells. By screening for C. hirsuta mutants with less lignified fruit valves, we found that loss of function of the transcription factor gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 7 (SPL7) caused a reduction in endocarpb cell-wall lignification and a consequent reduction in seed dispersal range. SPL7 is a conserved regulator of copper homeostasis and is both necessary and sufficient for copper to accumulate in the fruit. Laccases are copper-requiring enzymes. We discovered that laccase activity in endocarpb cell walls depends on the SPL7 pathway to acclimate to copper deficiency and provide sufficient copper for lignin polymerization. Hence, SPL7 links mineral nutrition to efficient dispersal of the next generation.

SQUINT Positively Regulates Resistance to the Pathogen Botrytis cinerea via miR156-SPL9 Module in Arabidopsis.

SQUINT (SQN) regulates plant maturation by promoting the activity of miR156, which functions primarily in the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) module regulating plant growth and development. Here, we show that SQN acts in the jasmonate (JA) pathway, a major signaling pathway regulating plant responses to insect herbivory and pathogen infection. Arabidopsis thaliana sqn mutants showed elevated sensitivity to the necrotrophic fungus Botrytis cinerea compared with wild type. However, SQN is not involved in the early pattern-triggered immunity response often triggered by fungal attack. Rather, SQN positively regulates the JA pathway, as sqn loss-of-function mutants treated with B. cinerea showed reduced JA accumulation, JA response and sensitivity to JA. Furthermore, the miR156-SPL9 module regulates plant resistance to B. cinerea: mir156 mutant, and SPL9 overexpression plants displayed elevated sensitivity to B. cinerea. Moreover, constitutively expressing miR156a or reducing SPL9 expression in the sqn-1 mutant restored the sensitivity of Arabidopsis to B. cinerea and JA responses. These results suggest that SQN positively modulates plant resistance to B. cinerea through the JA pathway, and the miR156-SPL9 module functions as a bridge between SQN and JA to mediate plant resistance to this pathogen.

SPL9 mediates freezing tolerance by directly regulating the expression of CBF2 in Arabidopsis thaliana

BackgroundFreezing stress inhibits plant development and causes significant damage to plants. Plants therefore have evolved a large amount of sophisticated mechanisms to counteract freezing stress by adjusting their growth and development correspondingly. Plant ontogenetic defense against drought, high salt, and heat stresses, has been extensively studied. However, whether the freezing tolerance is associated with ontogenetic development and how the freezing signals are delivered remain unclear.ResultsIn this study, we found that the freezing tolerance was increased with plant age at the vegetative stage. The expressions of microRNA156 (miR156) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), playing roles in regulation of ontogenetic development, were induced by cold stress. Overexpression of SPL9 (rSPL9) promoted the expression of C-REPEAT BINDING FACTOR 2 (CBF2) and hereafter enhanced the freezing tolerance. Genetic analysis indicated that the effect of rSPL9 on freezing tolerance is partially restored by cbf2 mutant. Further analysis confirmed that SPL9 directly binds to the promoter of CBF2 to activate the expression of CBF2, and thereafter increased the freezing tolerance.ConclusionsTherefore, our study uncovers a new role of SPL9 in fine-tuning CBF2 expression and thus mediating freezing tolerance in plants, and implies a role of miR156-SPL pathway in balancing the vegetative development and freezing response in Arabidopsis.

Overexpression of CpWRKY75 from Chimonanthus praecox Promotes Flowering Time in Transgenic Arabidopsis.

WRKY transcription factors play critical roles in the physiological processes of plants. Although the roles of WRKYs have been characterized in some model plants, their roles in woody plants, especially wintersweet (Chimonanthus praecox), are largely unclear. In this study, a wintersweet WRKY gene named CpWRKY75 belonging to group IIc was isolated and its characteristics were identified. CpWRKY75 is a nucleus-localized protein, and exhibited no transcriptional activation activity in yeast. CpWRKY75 was highly expressed in flowers at different bloom stages. Ectopic expression of CpWRKY75 significantly promoted the flowering time of transgenic Arabidopsis (Arabidopsis thaliana), as determined by the rosette leaf number and first flower open time. The expression levels of flowering-related genes were quantified by qRT-PCR, and the results suggested that CpWRKY75 had obvious influence on the expression level of MICRORNA156C (MIR156C), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), FLOWERING LOCUS T (FT), LEAFY (LFY), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), APETALA1 (AP1), CAULIFLOWER (CAL), and FRUITFULL (FUL). These results suggest that CpWRKY75 might have a flowering time regulation function, and additionally provide a new gene resource for the genetic engineering of woody flowering plants.

MeSPL9 attenuates drought resistance by regulating JA signaling and protectant metabolite contents in cassava.

Analysis of drought-related genes in cassava shows the involvement of MeSPL9 in drought stress tolerance and overexpression of a dominant-negative form of this gene demonstrates its negative roles in drought stress resistance. Drought stress severely impairs crop yield and is considered a primary threat to food security worldwide. Although the SQUAMOSA promoter binding protein-like 9 (SPL9) gene participates extensively in numerous developmental processes and in plant response to abiotic stimuli, its role and regulatory pathway in cassava (Manihot esculenta) response to the drought condition remain elusive. In the current study, we show that cassava SPL9 (MeSPL9) plays negative roles in drought stress resistance. MeSPL9expression was strongly repressed by drought treatment. Overexpression of a dominant-negative form of miR156-resistant MeSPL9, rMeSPL9-SRDX, in which a 12-amino acid repressor sequence was fused to rMeSPL9 at the C terminus, conferred drought tolerance without penalizing overall growth. rMeSPL9-SRDX-overexpressing lines not only exhibited increased osmoprotectant metabolites including proline and anthocyanin, but also accumulated more endogenous jasmonic acid (JA) and soluble sugars. Transcriptomic and real-time PCR analysis suggested that differentially expressed genes were involved in sugar or JA biosynthesis, signaling, and metabolism in transgenic cassava under drought conditions. Exogenous application of JA further confirmed that JA conferred improved drought resistance and promoted stomatal closure in cassava leaves. Taken together, our findings suggest that MeSPL9 affects drought resistance by modulating protectant metabolite levels and JA signaling, which have substantial implications for engineering drought tolerant crops.

ASCORBATE PEROXIDASE6 delays the onset of age-dependent leaf senescence.

Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs.

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions.

Do Arabidopsis Squamosa promoter binding Protein-Like genes act together in plant acclimation to copper or zinc deficiency?

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)‐dependent proteins, which includes enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu‐deficient growth conditions, Squamosa promoter binding Protein‐Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems, and it mediates a metabolic reorganization to economize on Cu. The transcription factor SPL7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by miRNA156 with roles in plant development. SPL7 shares extended regions of sequence homology with SPL1 and SPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with both the wild type and the spl7 single mutant under normal and Cu‐deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL7, but provided no indication for important roles of SPL1 or SPL12 in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn‐deficient wild type plants. Known Zn deficiency responses were unaltered in spl7, spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZnSOD activity is strongly downregulated in Zn‐deficient A. thaliana, in association with an about 94% reduction in the abundance of the CSD2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL7 and not associated with an upregulation of MIR398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 of Chlamydomonas. In the future, resolving how the specificity of SPL protein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of different SPL family proteins in the regulation of either Cu deficiency responses or growth and development of land plants.

Gibberellin repression of axillary bud formation in Arabidopsis by modulation of DELLA-SPL9 complex activity.

The formation of lateral branches has an important and fundamental contribution to the remarkable developmental plasticity of plants, which allows plants to alter their architecture to adapt to the challenging environment conditions. The Gibberellin (GA) phytohormones have been known to regulate the outgrowth of axillary meristems (AMs), but the specific molecular mechanisms remain unclear. Here we show that DELLA proteins regulate axillary bud formation by interacting and regulating the DNA-binding ability of SQUAMOSA-PROMOTER BINDING PROTEIN LIKE 9 (SPL9), a microRNA156-targeted squamosa promoter binding protein-like transcription factor. SPL9 participates in the initial regulation of axillary buds by repressing the expression of LATERAL SUPPRESSOR (LAS), a key regulator in the initiation of AMs, and LAS contributes to the specific expression pattern of the GA deactivation enzyme GA2ox4, which is specifically expressed in the axils of leaves to form a low-GA cell niche in this anatomical region. Nevertheless, increasing GA levels in leaf axils by ectopically expressing the GA-biosynthesis enzyme GA20ox2 significantly impaired axillary meristem initiation. Our study demonstrates that DELLA-SPL9-LAS-GA2ox4 defines a core feedback regulatory module that spatially pattern GA content in the leaf axil and precisely control the axillary bud formation in different spatial and temporal.

SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen in Arabidopsis thaliana.

SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen even with the sufficient level of copper in Arabidopsis thaliana. Under copper (Cu) deficiency, accumulation of mRNA encoding two Cu/Zn superoxide dismutases, CSD1 and CSD2, is downregulated to save Cu for plastocyanin. This downregulation depends on miR398 and is under the control of SQUAMOSA promoter-binding protein-like7 (SPL7). Arabidopsis seedlings are routinely cultured on Murashige and Skoog medium. However, the high nitrogen (N) content of the medium (60mM) has been shown to induce a similar response to Cu deficiency. The mRNA and protein levels of CSD1 and CSD2 are reduced under high N conditions, even if the Cu concentration in the medium is sufficient (0.1-0.5µM). In this study, we show that this symptom, similar to the Cu deficiency, occurred in the presence of high N largely depending on SPL7, suggesting that plants actually sensed Cu deficiency. However, a change in N concentration in the medium did not influence the total Cu concentration in shoots or roots. High N did not increase the protein content in leaves but facilitated rapid seedling growth. We speculate that this rapid growth causes a continuous Cu deficiency mainly because of high Cu uptake by mesophyll cells in the leaves. This idea was supported by the observation that plastocyanin did not overaccumulate at the range of 0.1-0.5µM Cu with 30mMN. In contrast, in the presence of 5µM Cu with 30mMN, plants accumulate more Cu in plastocyanin in the thylakoid lumen, resulting in a slight Cu deficiency in the chloroplast stroma. This process is independent of SPL7.

The MED30 subunit of mediator complex is essential for early plant development and promotes flowering in Arabidopsis thaliana.

Mediator is a large multiprotein complex that is required for the transcription of most, if not all, genes transcribed by RNA Polymerase II. A core set of subunits is essential to assemble a functional Mediator in vitro and, therefore, the corresponding loss-of-function mutants are expected to be lethal. The MED30 subunit is essential in animal systems, but is absent in yeast. Here, we report that MED30 is also essential for both male gametophyte and embryo development in the model plant Arabidopsis thaliana Mutant med30 pollen grains were viable and some were able to germinate and target the ovules, although the embryos aborted shortly after fertilization, suggesting that MED30 is important for the paternal control of early embryo development. When gametophyte defects were bypassed by specific pollen complementation, loss of MED30 led to early embryo development arrest. Later in plant development, MED30 promotes flowering through multiple signaling pathways; its downregulation led to a phase change delay, downregulation of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), FLOWERING LOCUS T (FTI) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and upregulation of FLOWERING LOCUS C (FLC).

Nitrate acts at the Arabidopsis thaliana shoot apical meristem to regulate flowering time.

Summary Optimal timing of flowering, a major determinant for crop productivity, is controlled by environmental and endogenous cues. Nutrients are known to modify flowering time; however, our understanding of how nutrients interact with the known pathways, especially at the shoot apical meristem (SAM), is still incomplete. Given the negative side‐effects of nitrogen fertilization, it is essential to understand its mode of action for sustainable crop production.We investigated how a moderate restriction by nitrate is integrated into the flowering network at the SAM, to which plants can adapt without stress symptoms.This condition delays flowering by decreasing expression of SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) at the SAM. Measurements of nitrate and the responses of nitrate‐responsive genes suggest that nitrate functions as a signal at the SAM. The transcription factors NIN‐LIKE PROTEIN 7 (NLP7) and NLP6, which act as master regulators of nitrate signaling by binding to nitrate‐responsive elements (NREs), are expressed at the SAM and flowering is delayed in single and double mutants. Two upstream regulators of SOC1 (SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE3 (SPL3) and SPL5) contain functional NREs in their promoters.Our results point at a tissue‐specific, nitrate‐mediated flowering time control in Arabidopsis thaliana.

Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications.

Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.