The CUL3A-LFH1-UBC15 ubiquitin ligase complex mediates SHORT VEGETATIVE PHASE degradation to accelerate flowering at high ambient temperature.

Ambient temperature affects flowering time in plants, and the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a crucial role in the response to changes in ambient temperature. SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature, but the details of SVP degradation are unclear. Here, we show that SVP degradation at high ambient temperature is mediated by the CULLIN3-RING E3 ubiquitin ligase (CRL3) complex in Arabidopsis thaliana. We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain. We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1 (LFH1). Single mutants of LFH1 or CULLIN3A (CUL3A) showed late flowering specifically at 27°C. LFH1 protein levels increased at high ambient temperature. We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP-CUL3A complex formation. Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature, suggesting that the CUL3-LFH1 complex functions in SVP degradation. Screening E2 ubiquitin-conjugating enzymes (UBCs) using RING-BOX PROTEIN 1 (RBX1), a component of the CRL3 complex, as bait identified UBC15. ubc15 mutants also showed late flowering at high ambient temperature. In vitro and in vivo ubiquitination assays using recombinant CUL3A, LFH1, RBX1, and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner. Collectively, these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A, LFH1, and UBC15.

Chloroplasts prevent precocious flowering through a GOLDEN2-LIKE-B-BOX DOMAIN PROTEIN module.

The timing of flowering is tightly controlled by signals that integrate environmental and endogenous cues. Sugars produced by carbon fixation in the chloroplast are a crucial endogenous cue for floral initiation. Chloroplasts also convey information directly to the nucleus through retrograde signaling to control plant growth and development. Here, we show that mutants defective in chlorophyll biosynthesis and chloroplast development flowered early, especially under long-day conditions, although low sugar accumulation was seen in some mutants. Plants treated with the bleaching herbicide norflurazon also flowered early, suggesting that chloroplasts have a role in floral repression. Among retrograde signaling mutants, the golden2-like 1 (glk1) glk2 double mutants showed early flowering under long-day conditions. This early flowering was completely suppressed by constans (co) and flowering locus t (ft) mutations. Leaf vascular-specific knockdown of both GLK1 and GLK2 phenocopied the glk1 glk2 mutants. GLK1 and GLK2 repress flowering by directly activating the expression of B-BOX DOMAIN PROTEIN 14 (BBX14), BBX15, and BBX16 via CCAATC cis-elements in the BBX genes. BBX14/15/16 physically interact with CO in the nucleus, and expression of BBXs hampered CO-mediated FT transcription. Simultaneous knockdown of BBX14/15/16 by artificial miRNA (35S::amiR-BBX14/15/16) caused early flowering with increased FT transcript levels, whereas BBX overexpression caused late flowering. Flowering of glk1/2 and 35S::amiR-BBX14/15/16 plants was insensitive to norflurazon treatment. Taking these observations together, we propose that the GLK1/2-BBX14/15/16 module provides a novel mechanism explaining how the chloroplast represses flowering to balance plant growth and reproductive development.

Profiling Protein-DNA Interactions by Chromatin Immunoprecipitation in Arabidopsis.

In plant cells, transcription factors play an important role in the regulation of gene expression, which eventually leads to the formation of complex phenotypes. Although chromatin immunoprecipitation (ChIP) involves a lengthy process that requires up to 4 days to complete, it is a powerful technique to investigate the interactions between transcription factors and their target sequences in vivo. Here, we describe a detailed ChIP protocol, focusing on ChIP-qPCR, from material collection to data analyses. Moreover, we explain multiple checkpoints for the quality control of ChIP-qPCR data to ensure the success of this protocol. As this protocol is robust, it can be adapted to other plant materials and plant species, and it can be used for genome-wide profiling experiments, including ChIP-chip and ChIP-seq analyses. We believe that our ChIP-qPCR protocol facilitates research on the interactions between plant transcription factors and their target sequences in vivo.

PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 modulates flowering in a florigen-independent manner by regulating SVP.

PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 (PECT1) regulates phosphatidylethanolamine biosynthesis and controls the phosphatidylethanolamine:phosphatidylcholine ratio in Arabidopsis thaliana Previous studies have suggested that PECT1 regulates flowering time by modulating the interaction between phosphatidylcholine and FLOWERING LOCUS T (FT), a florigen, in the shoot apical meristem (SAM). Here, we show that knockdown of PECT1 by artificial microRNA in the SAM (pFD::amiR-PECT1) accelerated flowering under inductive and even non-inductive conditions, in which FT transcription is almost absent, and in ft-10 twin sister of ft-1 double mutants under both conditions. Transcriptome analyses suggested that PECT1 affects flowering by regulating SHORT VEGETATIVE PHASE (SVP) and GIBBERELLIN 20 OXIDASE 2 (GA20ox2). SVP misexpression in the SAM suppressed the early flowering of pFD::amiR-PECT1 plants. pFD::amiR-PECT1 plants showed increased gibberellin (GA) levels in the SAM, concomitant with the reduction of REPRESSOR OF GA1-3 levels. Consistent with this, GA treatment had little effect on flowering time of pFD::amiR-PECT1 plants and the GA antagonist paclobutrazol strongly affected flowering in these plants. Together, these results suggest that PECT1 also regulates flowering time through a florigen-independent pathway, modulating SVP expression and thus regulating GA production.

Arabidopsis ABF3 and ABF4 Transcription Factors Act with the NF-YC Complex to Regulate SOC1 Expression and Mediate Drought-Accelerated Flowering.

The drought-escape response accelerates flowering in response to drought stress, allowing plants to adaptively shorten their life cycles. Abscisic acid (ABA) mediates plant responses to drought, but the role of ABA-responsive element (ABRE)-binding factors (ABFs) in the drought-escape response is poorly understood. Here, we show that Arabidopsis thaliana ABF3 and ABF4 regulate flowering in response to drought through transcriptional regulation of the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). The abf3 abf4 mutant displayed ABA-insensitive late flowering under long-day conditions. Ectopic expression of ABF3 or ABF4 in the vasculature, but not in the shoot apex, induced early flowering, whereas expression of ABF3 fused with the SRDX transcriptional repressor domain delayed flowering. We identified SOC1 as a direct downstream target of ABF3/4, and found that SOC1 mRNA levels were lower in abf3 abf4 than in wild-type plants. Moreover, induction of SOC1 by ABA was hampered in abf3 abf4 mutants. ABF3 and ABF4 were enriched at the -1028- to -657-bp region of the SOC1 promoter, which does not contain canonical ABF-ABRE-binding motifs but has the NF-Y binding element. We found that ABF3 and ABF4 interact with nuclear factor Y subunit C (NF-YC) 3/4/9 in vitro and in planta, and induction of SOC1 by ABA was hampered in nf-yc3 yc4 yc9 mutants. Interestingly, the abf3 abf4, nf-yc3 yc4 yc9, and soc1 mutants displayed a reduced drought-escape response. Taken together, these results suggest that ABF3 and ABF4 act with NF-YCs to promote flowering by inducing SOC1 transcription under drought conditions. This mechanism might contribute to adaptation by enabling plants to complete their life cycles under drought stress.

CC-type glutaredoxins mediate plant response and signaling under nitrate starvation in Arabidopsis.

BACKGROUND: Nitrogen is an essential nutrient in plants. Despite the importance of nitrogen for plant growth and agricultural productivity, signal transduction pathways in response to nitrate starvation have not been fully elucidated in plants. RESULTS: Gene expression analysis and ectopic expression were used to discover that many CC-type glutaredoxins (ROXYs) are differentially expressed in response to nitrate deprivation. A gain-of-function approach showed that ROXYs may play a role in nutrient sensing through the regulation of chlorophyll content, root hair growth, and transcription of nitrate-related genes such as NRT2.1 under low or high nitrate conditions. Reactive oxygen species (ROS) were produced in plant roots under nitrate starvation and H2O2 treatment differentially regulated the expression of the ROXYs, suggesting the involvement of ROS in signaling pathways under nitrate deficiency. CONCLUSION: This work adds to what is known about nitrogen sensing and signaling through the findings that the ROXYs and ROS are likely to be involved in the nitrate deprivation signaling pathway.

Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain.

Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input.

PHO1 Exports Phosphate from the Chalazal Seed Coat to the Embryo in Developing Arabidopsis Seeds.

Seed production requires the transfer of nutrients from the maternal seed coat to the filial endosperm and embryo. Because seed coat and filial tissues are symplasmically isolated, nutrients arriving in the seed coat via the phloem must be exported to the apoplast before reaching the embryo. Proteins implicated in the transfer of inorganic phosphate (Pi) from the seed coat to the embryo are unknown despite seed P content being an important agronomic trait. Here we show that the Arabidopsis Pi exporters PHO1 and PHOH1 are expressed in the chalazal seed coat (CZSC) of developing seeds. PHO1 is additionally expressed in developing ovules. Phosphorus (P) content and Pi flux between the seed coat and embryo were analyzed in seeds from grafts between WT roots and scions from either pho1, phoh1, or the pho1 phoh1 double mutant. Whereas P content and distribution between the seed coat and embryo in fully mature dry seeds of these mutants are similar to the WT, at the mature green stage of seed development the seed coat of the pho1 and pho1 phoh1 mutants, but not of the phoh1 mutant, retains approximately 2-fold more P than its WT control. Expression of PHO1 under a CZSC-specific promoter complemented the seed P distribution phenotype of the pho1 phoh1 double mutant. CZSC-specific down-expression of PHO1 also recapitulated the seed P distribution phenotype of pho1. Together, these experiments show that PHO1 expression in the CZSC is important for the transfer of P from the seed coat to the embryo in developing seeds.

SCYL2 Genes Are Involved in Clathrin-Mediated Vesicle Trafficking and Essential for Plant Growth.

Protein transport between organelles is an essential process in all eukaryotic cells and is mediated by the regulation of processes such as vesicle formation, transport, docking, and fusion. In animals, SCY1-LIKE2 (SCYL2) binds to clathrin and has been shown to play roles in trans-Golgi network-mediated clathrin-coated vesicle trafficking. Here, we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis (Arabidopsis thaliana) homologs of animal SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 isoforms using multiple single or double loss-of-function alleles show that SCYL2B is involved in root hair development and that SCYL2A and SCYL2B are essential for plant growth and development and act redundantly in those processes. Quantitative reverse transcription-polymerase chain reaction and a ß-glucuronidase-aided promoter assay show that SCYL2A and SCYL2B are differentially expressed in various tissues. We also show that SCYL2 proteins localize to the Golgi, trans-Golgi network, and prevacuolar compartment and colocalize with Clathrin Heavy Chain1 (CHC1). Furthermore, bimolecular fluorescence complementation and coimmunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble NSF Attachment Protein Receptors (SNAREs): Vesicle Transport through t-SNARE Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip localization of Cellulose Synthase-Like D3 is dependent on SCYL2B. These findings suggest the role of SCYL2 genes in plant cell developmental processes via clathrin-mediated vesicle membrane trafficking.

Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.

Phosphorus is a macronutrient taken up by cells as inorganic phosphate (P(i)). How cells sense cellular P(i) levels is poorly characterized. Here, we report that SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to P(i) availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and P(i) transport in Arabidopsis In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate P(i) starvation responses. We propose that InsPs communicate cytosolic P(i) levels to SPX domains and enable them to interact with a multitude of proteins to regulate P(i) uptake, transport, and storage in fungi, plants, and animals.

The EXS Domain of PHO1 Participates in the Response of Shoots to Phosphate Deficiency via a Root-to-Shoot Signal.

The response of shoots to phosphate (Pi) deficiency implicates long-distance communication between roots and shoots, but the participating components are poorly understood. We have studied the topology of the Arabidopsis (Arabidopsis thaliana) PHOSPHATE1 (PHO1) Pi exporter and defined the functions of its different domains in Pi homeostasis and signaling. The results indicate that the amino and carboxyl termini of PHO1 are both oriented toward the cytosol and that the protein spans the membrane twice in the EXS domain, resulting in a total of six transmembrane α-helices. Using transient expression in Nicotiana benthamiana leaf, we demonstrated that the EXS domain of PHO1 is essential for Pi export activity and proper localization to the Golgi and trans-Golgi network, although the EXS domain by itself cannot mediate Pi export. In contrast, removal of the amino-terminal hydrophilic SPX domain does not affect the Pi export capacity of the truncated PHO1 in N. benthamiana. While the Arabidopsis pho1 mutant has low shoot Pi and shows all the hallmarks associated with Pi deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes, expression of only the EXS domain of PHO1 in the roots of the pho1 mutant results in a remarkable improvement of shoot growth despite low shoot Pi. Transcriptomic analysis of pho1 expressing the EXS domain indicates an attenuation of the Pi signaling cascade and the up-regulation of genes involved in cell wall synthesis and the synthesis or response to several phytohormones in leaves as well as an altered expression of genes responsive to abscisic acid in roots.

Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

Ethylene mediates response and tolerance to potassium deprivation in Arabidopsis.

Potassium deprivation leads to large reductions in plant growth and yields. How plants sense and transduce the stress signals initiated by potassium deprivation is poorly understood. Both ethylene production and the transcription of genes involved in ethylene biosynthesis increase when plants are deprived of potassium. To elucidate the role of ethylene in low potassium signaling pathways, we used both genetic and chemical approaches. Our results showed that ethylene is important in tolerance to low potassium and for changes in both root hair and primary root growth in Arabidopsis thaliana. We show that ethylene acts upstream of reactive oxygen species in response to potassium deprivation. The expression of High-Affinity K(+) Transporter5 was used as a marker of potassium deprivation and was found to be dependent on ethylene signaling. In the ethylene insensitive2-1 (ein2-1) mutant, the ethylene-mediated low potassium responses were not completely eliminated, suggesting that some potassium deprivation-induced responses are either ethylene independent or EIN2 independent. Ethylene signaling is a component of the plant's response to low potassium that stimulates the production of reactive oxygen species and is important for changes in root morphology and whole plant tolerance to low potassium conditions.

A role for phosphatidylinositol 3-phosphate in abscisic acid-induced reactive oxygen species generation in guard cells.

Guard cells generate reactive oxygen species (ROS) in response to abscisic acid (ABA), which leads to stomatal closing. The upstream steps of the ABA-induced ROS generation pathway remain largely unknown. In animal cells, ROS generation in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P). Stomatal guard cells contain PI3P and PI 3-kinase activity. In this study, we tested whether PI3P has a role in ROS generation in guard cells exposed to ABA. We found that PI 3-kinase inhibitors wortmannin or LY294002 inhibited ABA-induced ROS generation and stomatal closing. Endosome-binding domain (of human EEA1), which specifically binds to PI3P, also inhibited ABA-induced ROS generation and stomatal closing when overexpressed in guard cells. Hydrogen peroxide partially reversed the effects of wortmannin or LY294002 on ABA-induced stomatal closing. These results support a role for PI3P in ABA-induced ROS generation and stomatal closing movement.

Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements.

Phosphatidylinositol (PI) metabolism plays a central role in signaling pathways in both animals and higher plants. Stomatal guard cells have been reported to contain PI 3-phosphate (PI3P) and PI 4-phosphate (PI4P), the products of PI 3-kinase (PI3K) and PI 4-kinase (PI4K) activities. In this study, we tested the roles of PI3P and PI4P in stomatal movements. Both wortmannin (WM) and LY294002 inhibited PI3K and PI4K activities in guard cells and promoted stomatal opening induced by white light or the circadian clock. WM and LY294002 also inhibited stomatal closing induced by abscisic acid (ABA). Furthermore, overexpression in guard cells of GFP:EBD (green fluorescent protein:endosome binding domain of human EEA1) or GFP:FAPP1PH (PI-four-P adaptor protein-1 pleckstrin homology domain), which bind to PI3P and PI4P, respectively, increased stomatal apertures under darkness and white light and partially inhibited stomatal closing induced by ABA. The reduction in ABA-induced stomatal closing with reduced levels of PI monophosphate seemed to be attributable, at least in part, to impaired Ca(2+) signaling, because WM and LY294002 inhibited ABA-induced cytosolic Ca(2+) increases in guard cells. These results suggest that PI3P and PI4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PI3P and PI4P enhance stomatal opening.