Summary
Flowering time is a relevant agronomic trait because is crucial for the optimal formation of seeds and fruits. The genetic pathways controlling this developmental phase transition have been ...studied extensively in Arabidopsis thaliana. These pathways converge in a small number of genes including FT, the so‐called florigen, which integrates environmental cues like ambient temperature. Nevertheless, detailed and functional studies about flowering time in Brassica crops are scarce. Here we study the role of the FT Brassica rapa homologues and the effect of high ambient temperature on flowering time in this crop. Phenotypic characterization and gene‐expression analyses suggest that BraA.FT.a (BraA02g016700.3C) is decisive for initiating floral transition; consequently, braA.ft.a loss‐of‐function and hypomorphic mutations result in late flowering phenotypes. We also show that high ambient temperature delays B. rapa floral transition by reducing BraA.FT.a expression. Strikingly, these expression changes are associated with increased histone H2A.Z levels and less accessible chromatin configuration of the BraA.FT.a locus at high ambient temperature. Interestingly, increased H2A.Z levels at high ambient temperature were also observed for other B. rapa temperature‐responsive genes. Previous reports delimited that Arabidopsis flowers earlier at high ambient temperature due to reduced H2A.Z incorporation in the FT locus. Our data reveal a conserved chromatin‐mediated mechanism in B. rapa and Arabidopsis in which the incorporation of H2A.Z at FT chromatin in response to warm ambient temperature results in different flowering time responses. This work will help to develop improved Brassica crop varieties with flowering time requirements to cope with global warming.
Open Research Badges
This article has earned an Open Materials Badge for making publicly available the components of the research methodology needed to reproduce the reported procedure and analysis. Methods are available at protocols.iodx.doi.org/10.17504/protocols.io.zmff43n.
Significance Statement
Flowering time is a key agronomic trait that responds to moderate changes in ambient temperature. We found that high ambient temperature modulates Brassica rapa flowering time, altering the chromatin composition of BraA.FT.a locus differently from its orthologue Arabidopsis thaliana FT locus. Although in both species a role for H2A.Z is conserved in response to warmth, we uncovered unexpected H2A.Z‐nucloseome differences in behaviour between these model and crop Brassica species.
Knowledge concerning the integration of genetic pathways mediating the responses to environmental cues controlling flowering initiation in crops is scarce. Here, we reveal the diversity in oilseed ...rape (OSR) flowering response to high ambient temperature. Using a set of different spring OSR varieties, we found a consistent flowering delay at elevated temperatures. Remarkably, one of the varieties assayed exhibited the opposite behaviour. Several FT‐like paralogs are plausible candidates to be part of the florigen in OSR. We revealed that BnaFTA2 plays a major role in temperature‐dependent flowering initiation. Analysis of the H2A.Z histone variant occupancy at this locus in different Brassica napus varieties produced contrasting results, suggesting the involvement of additional molecular mechanisms in BnaFTA2 repression at high ambient temperature. Moreover, BnARP6 RNAi plants showed little accumulation of H2A.Z at high temperature while maintaining temperature sensitivity and delayed flowering. Furthermore, we found that H3K4me3 present in BnaFTA2 under inductive flowering conditions is reduced at high temperature, suggesting a role for this hallmark of transcriptionally active chromatin in the OSR flowering response to warming. Our work emphasises the plasticity of flowering responses in B. napus and offers venues to optimise this process in crop species grown under suboptimal environmental conditions.
Summary statement
Ambient temperature is critical to set the timing for flowering and farming practices. We have uncovered variability in flowering time in spring varieties of oilseed rape (OSR) under high ambient temperature and determined their transcriptomic profiles. We concluded that warm temperature delays flowering in these varieties by decreasing the expression of FT homologues, being BnaFTA2 a key candidate in temperature‐dependent flowering control. Both H2A.Z‐dependent and independent mechanisms control OSR flowering time under high ambient temperatures.
Root knot nematodes (RKNs) penetrate into the root vascular cylinder, triggering morphogenetic changes to induce galls, de novo formed ‘pseudo-organs’ containing several giant cells (GCs). ...Distinctive gene repression events observed in early gall/GCs development are thought to be mediated by post-transcriptional silencing via microRNAs (miRNAs), a process that is far from being fully characterized.
Arabidopsis thaliana backgrounds with altered activities based on target 35S:: MIMICRY172 (MIM172), 35S::TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1)-miR172-resistant (35S::TOE1
R) and mutant (flowering locus T-10 (ft-10)) lines were used for functional analysis of nematode infective and reproductive parameters. The GUS-reporter lines, MIR172A–E::GUS, treated with auxin (IAA) and an auxin-inhibitor (a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA)), together with the MIR172C AuxRE::GUS line with two
mutated auxin responsive elements (AuxREs), were assayed for nematode-dependent gene expression.
Arabidopsis thaliana backgrounds with altered expression of miRNA172, TOE1 or FT showed lower susceptibility to the RKNs and smaller galls and GCs. MIR172C–D::GUS showed restricted promoter activity in galls/GCs that was regulated by auxins through auxin-responsive factors. IAA induced their activity in galls while PEO-IAA treatment and mutations in AuxRe motifs abolished it.
The results showed that the regulatory module miRNA172/TOE1/FT plays an important role in correct GCs and gall development, where miRNA172 is modulated by auxins.
Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is ...viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells.
Fatty acids have been recognized as regulators of immune function in addition to their known metabolic role. Long-chain fatty acids bind free fatty acid receptor (FFAR)-1/GPR40, which is expressed on ...bovine neutrophils, and increase responses such as granule release and gene expression. In this study, we investigated the molecular mechanisms governing the up-regulation of cyclooxygenase-2 (COX-2) and IL-8, as well as matrix metalloproteinase (MMP)-9 granule release in FFAR1/GPR40 agonist-stimulated neutrophils. Our results showed that natural (oleic and linoleic acid) and synthetic (GW9508) FFAR1/GPR40 agonists increased ERK1/2, p38 MAPK and Akt phosphorylation, and that the FFAR1/GPR40 antagonist GW1100 reduced these responses. We evaluated the levels of IκBα, a component of the classical activation pathway of the transcription factor NF-κB, and we observed IκBα reduction after stimulation with FFAR1/GPR40 agonists, an effect that was inhibited by GW1100 or the inhibitors UO126, SB203580 or LY294002. FFAR1/GPR40 agonists increased COX-2 and IL-8 expression, which was inhibited by GW1100 and an NF-κB inhibitor. Finally, the FFAR1/GPR40 agonist-induced MMP-9 granule release was reduced by GW1100 and UO126. In conclusion, FFAR1/GPR40 agonists differentially stimulate neutrophil functions; COX-2 and IL-8 are expressed after FFAR1/GPR40 activation via NF-κB, IκBα reduction is FFAR1/GPR40- and PI3K/MAPK-dependent, and MMP-9 granule release is FFAR1/GPR40- and ERK1/2-dependent.
A combination of cost effective sublethal
Daphnia magna feeding tests, yeast- and cell culture-based bioassays and Toxicity Identification Evaluation (TIE) procedures was used to characterize toxic ...compounds within sediments collected in a river area under the influence of the effluents from a chlor-alkali industry (Ebro River, NE Spain). Tests were designed to measure and identify toxic compounds in the particulate and filtered water fractions of sediment elutriates. The combined use of bioassays responding to elutriates and dioxin-like compounds evidenced the existence of three major groups of hazardous contaminants in the most contaminated site: (A) metals such as cadmium and mercury bound to sediment fine particles that could be easily resuspended and moved downstream, (B) soluble compounds (presumably, lye) able to alkalinize water to toxic levels, and (C) organochlorine compounds with high dioxin-like activity. These results provided evidence that elutriate
D. magna feeding responses can be used as surrogate assays for more tedious chronic whole sediment tests, and that the incorporation of such tests in sediment TIE procedures may improve the ability to identify the toxicity of particle-bound and water-soluble contaminants in sediments.
Aquatic organisms are often exposed to mixtures of low levels of pollutants whose presence and effects can pass easily unnoticed if only traditional monitoring strategies are employed. The main aim ...of this work was to assess the presence and effects of trace levels of pollutants in a scarcely affected area through the combination of chemical and biological approaches. Sediments were collected along a river with little anthropogenic pressure and assayed for cytochrome P450 (Cyp1a)-dependent ethoxyresorufin-
O
-deethylase (EROD) activity with the rainbow trout gonadal cell line RTG-2. Chemical analyses were performed in these sediments using two-dimensional gas chromatography-time-of-flight mass spectrometry. Sediment samples induced EROD activity, and chemical analyses evidenced the presence of a wide variety of contaminants in the range of nanograms per gram of dry weight. Correlation analysis between EROD induction and chemical analyses data showed an
r
value of 0.840 (
p
<
0.05). In addition, fish from a fish farm located downstream of the sampling points exhibited high hepatic EROD levels as well as an induced expression of
cyp1a
and
cyp3a
. In conclusion, only an appropriate combination of biological and chemical techniques allowed the detection of the presence of trace levels of contaminants in a theoretically nonaffected river.
Summary
Pollen development is a crucial step in higher plants, which not only makes possible plant fertilization and seed formation, but also determines fruit quality and yield in crop species. Here, ...we reported a tomato T‐DNA mutant, pollen deficient1 (pod1), characterized by an abnormal anther development and the lack of viable pollen formation, which led to the production of parthenocarpic fruits. Genomic analyses and the characterization of silencing lines proved that pod1 mutant phenotype relies on the tomato SlMED18 gene encoding the subunit 18 of Mediator multi‐protein complex involved in RNA polymerase II transcription machinery. The loss of SlMED18 function delayed tapetum degeneration, which resulted in deficient microspore development and scarce production of viable pollen. A detailed histological characterization of anther development proved that changes during microgametogenesis and a significant delay in tapetum degeneration are associated with a high proportion of degenerated cells and, hence, should be responsible for the low production of functional pollen grains. Expression of pollen marker genes indicated that SlMED18 is essential for the proper transcription of a subset of genes specifically required to pollen formation and fruit development, revealing a key role of SlMED18 in male gametogenesis of tomato. Additionally, SlMED18 is able to rescue developmental abnormalities of the Arabidopsis med18 mutant, indicating that most biological functions have been conserved in both species.
Significance Statement
Pollination is a key development process in the life cycle of flowering plants. Genetic and molecular characterization of a tomato mutant have led to the identification of POD1 gene encoding the Mediator complex subunit MED18 whose function is required for tapetum tissue degeneration, a crucial step for pollen development. Furthermore, we show that MED18 fulfils an essential role in tomato, ensuring proper gene regulation during pollen ontogeny.
Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells ...originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types.
Summary
We have characterized a mutation affecting the Arabidopsis EARLY IN SHORT DAYS 7 (ESD7) gene encoding the catalytic subunit of DNA polymerase epsilon (ε), AtPOL2a. The esd7‐1 mutation causes ...early flowering independently of photoperiod, shortened inflorescence internodes and altered leaf and root development. esd7‐1 is a hypomorphic allele whereas knockout alleles displayed an embryo‐lethal phenotype. The esd7 early flowering phenotype requires functional FT and SOC1 proteins and might also be related to the misregulation of AG and AG‐like gene expression found in esd7. Genes involved in the modulation of chromatin structural dynamics, such as LHP1/TFL2 and EBS, which negatively regulate FT expression, were found to interact genetically with ESD7. In fact a molecular interaction between the carboxy terminus of ESD7 and TFL2 was demonstrated in vitro. Besides, fas2 mutations suppressed the esd7 early flowering phenotype and ICU2 was found to interact with ESD7. Discrete regions of the chromatin of FT and AG loci were enriched in activating epigenetic marks in the esd7‐1 mutant. We concluded that ESD7 might be participating in processes involved in chromatin‐mediated cellular memory.
PDF
