One of the greatest threats to wild strawberries (
Fragaria vesca
Mara des Bois) after harvest is the highly perishability at ambient temperature. Breeders have successfully met the quality demands ...of consumers, but the prevention of waste after harvest in fleshy fruits is still pending. Most of the waste is due to the accelerated progress of senescence-like process after harvest linked to a rapid loss of water and firmness at ambient temperature. The storage life of strawberries increases at low temperature, but their quality is limited by the loss of cell structure. The application of high CO
2
concentrations increased firmness during cold storage. However, the key genes related to resistance to softening and cell wall disassembly following transference from cold storage at 20°C remain unclear. Therefore, we performed RNA-seq analysis, constructing a weighted gene co-expression network analysis (WGCNA) to identify which molecular determinants play a role in cell wall integrity, using strawberries with contrasting storage conditions, CO
2
-cold stored (CCS), air-cold stored (ACS), non-cold stored (NCS) kept at ambient temperature, and intact fruit at harvest (AH). The hub genes associated with the cell wall structural architecture of firmer CO
2
-treated strawberries revealed xyloglucans stabilization attributed mainly to a down-regulation of
Csl E1
,
XTH 15
,
Exp-like B1
and the maintenance of expression levels of nucleotide sugars transferases such as
GMP
and
FUT
as well as improved lamella integrity linked to a down-regulation of
RG-lyase
,
PL-like
and
PME
. The preservation of cell wall elasticity together with the up-regulation of
LEA
,
EXPA4
, and
MATE
, required to maintain cell turgor, is the mechanisms controlled by high CO
2
. In stressed air-cold stored strawberries, in addition to an acute softening, there is a preferential transcript accumulation of genes involved in lignin and raffinose pathways. Non-cold stored strawberries kept at 20°C after harvest are characterized by an enrichment in genes mainly involved in oxidative stress and up-expression of genes involved in jasmonate biosynthesis. The present results on transcriptomic analysis of CO
2
-treated strawberries with enhanced resistance to softening and oxidative stress at consumption will help to improve breeding strategies of both wild and cultivated strawberries.
Pathogen infections require the production of effectors that enable host colonization. Effectors have diverse functions and are only expressed at certain stages of the infection cycle. Thus, effector ...genes are tightly regulated by several mechanisms, including chromatin remodeling. Here, we investigate the role of histone acetylation in effector gene activation in the fungal wheat pathogen
. We demonstrate that lysine acetyltransferases (KATs) are essential for the spatiotemporal regulation of effector genes. We show that the KAT Sas3 is involved in leaf symptom development and pycnidia formation. Importantly, our results indicate that Sas3 controls histone acetylation of effector loci and is a regulator of effector gene activation during stomatal penetration. Overall, our work demonstrates the key role of histone acetylation in regulating gene expression associated with plant infection.
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
Sl
MED
18
gene encoding the subunit 18 of Mediator multi‐protein complex involved in
RNA
polymerase
II
transcription machinery. The loss of
Sl
MED
18
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
Sl
MED
18
is essential for the proper transcription of a subset of genes specifically required to pollen formation and fruit development, revealing a key role of
Sl
MED
18
in male gametogenesis of tomato. Additionally,
Sl
MED
18
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
POD
1
gene encoding the Mediator complex subunit
MED
18 whose function is required for tapetum tissue degeneration, a crucial step for pollen development. Furthermore, we show that
MED
18 fulfils an essential role in tomato, ensuring proper gene regulation during pollen ontogeny.
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 is thought to be mediated by post-transcriptional silencing
via
-miRNAs, a process far from being fully characterized.
Arabidopsis
backgrounds with altered activities based on target MIMICRY (
35S::MIM172
), 35S::TOE1-miR172-resistant (
35S::TOE1
R
) and mutant (
ft-10
) lines, were used for functional analysis of nematode infective and reproductive parameters. GUS-reporter lines,
MIR172A-E::GUS
, auxin (IAA) and auxin-inhibitor (PEO-IAA) treatments, as well as a
MIR172C
AuxRE−
::GUS
line with two mutated auxin responsive elements (
AuxREs
) were assayed for nematode-dependent gene expression.
Arabidopsis
backgrounds with altered expression of either MIRNA172,
TARGET OF EAT1
(
TOE1
), and
FLOWERING LOCUS T
(
FT
), showed lower susceptibility to the RKNs and smaller feeding sites, galls/GCs.
MIR172C-D::GUS
showed restricted activity in galls/GCs that is 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 show that the activity of the regulatory module
miRNA172/TOE1/FT
plays an important role in correct GCs and gall development where miRNA172 is modulated by auxins.
The annotation of human genomic diversity is nowadays at an unprecedented rapid pace, but arguably still limited by the lack of computational approaches that integrate the analysis of multiple forms ...of genetic variation and are suitable for the analysis of both, whole‐genome sequencing and targeted sequencing strategies. For the analysis of structural variations (SVs), for example, which include >50 bp deletions, duplications, copy‐number variations (CNVs), inversions, mobile‐element insertions, and translocations, only a few computational tools integrate breakpoint signatures and read‐depth information in a single approach. And, none of them makes use, in the analysis of targeted sequencing, of breakpoint information to filter false‐positive CNVs when small adjacent introns are completely captured despite that targeted sequencing is still more popular than whole‐genome sequencing (e.g. exome‐sequencing and many common approaches of genetic‐testing are forms of targeted sequencing). Here, we present the development of GRAPES (Genomic Rearrangement Analyzer for Paired‐End Sequencing), which is as a versatile tool that allows the analysis of SVs on whole‐genome and targeted DNA sequencing data by integrating discordant‐pairs, split‐reads, read‐depth, and local assembly. In the case of targeted sequencing, GRAPES uses on‐ and off‐ target read depth coupled with breakpoint analysis to provide accurate predictions. We have validated GRAPES using the accurately annotated NA12878 genome, the ICR96 CNV exon validation series, and a panel of simulated datasets, and it achieves higher rates of sensitivity and specificity than previously developed approaches. We have tested GRAPES to annotate SVs from targeted sequencing data of patients affected with inherited cardiac disorders, uncovering complex SVs that were unresolved by using other algorithms. In sum, GRAPES not only is a comprehensive tool for the analysis of SVs from whole‐genome sequencing and targeted sequencing data, but also allows the annotation of SVs overlooked by current single‐approach approaches.
Support or Funding Information
International Doctorate Grant in Biomedical Research and Public health of the Università Cattolica del Sacro Cuore
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Display omitted
•Restricted mobilization of sucrose in CO2-pretreated strawberries.•Up regulation of FvSS and FvCWINV at low temperatures.•Down-regulation of FvVINV2 expression and sucrose ...accumulation.•Up regulation of FvSPS1 expression in dark red strawberries.•Carbohydrate conservation in CO2-pretreated strawberries.
Sucrose is a key player in strawberry metabolism. However, the molecular mechanisms underlying sucrose status in strawberries during later ripening stages and postharvest storage remains quite limited. Therefore, we identified members of various gene families encoding key enzymes involved in sucrose metabolism, analyzed their expression profiles and determined the sugar content in strawberries at different ripening stages and postharvest treatments. Three different ripening stages around harvest time, almost red (AR), full red (FR) and dark red (DR) were analyzed, as well as after pre-treatment with high CO2 levels (18 % CO2, 2 d) during low temperature (LT) storage and further shelf-life at 20 °C. The accumulation of sucrose, hexoses, trehalose, myo-inositol, fructo-oligosaccharides (FOS) and raffinose family oligosaccharides (RFOs) was analyzed. Our results indicate that the transition from FR to DR stages was characterized by a prominent accumulation of hexoses, sucrose and a sharp decrease in the levels of FOS. We therefore suggest that FOS, in addition to their protective role in stressful conditions, are also important metabolic signals of the end of ripening process. The highest levels of RFOs was detected in strawberries at LT in air, making them a possible marker of damage, an idea reinforced too by the low RFOs and high myo-inositol quantities found in high CO2 protective treatment. Interestingly, the sucrose accumulation in fully ripe strawberries and in high CO2-treated ones was linked to a significant decrease in the expression of FvVIN2 (vacuolar invertase), an opposite trend to that observed in fruit stored in air. Furthermore, high levels of CO2 prevented both the upregulation of FvSS1 and the sharp increase in the expression of FvCWINV1 observed at low temperature in air. The efficiency of high CO2 pre-treatment on sucrose accumulation was particularly evident during the shelf-life period at 20 °C (SL) after LT storage. Our study provides new insights into how strawberries become sweet by retaining sucrose content. Additionally, our results clearly highlight the need to specify the sugar status at harvest to know the effectiveness of a treatment in maintaining quality attributes.
•Neutrophils express the free fatty acids (FFA) 4 receptor, which is activated by docosahexaenoic acid (DHA) and TUG-891.•DHA and TUG-891 increased matrix metalloproteinase (MMP)-9 granules release ...and superoxide production.•Intracellular calcium is key in FFA4 agonists-induced superoxide production.
Fatty acids are well known metabolic intermediaries but also have a role in the immune response. Long-chain fatty acids such as omega-6 and -9 activate neutrophil function through free fatty acid (FFA)-1 receptor in bovines. Although omega-3 has also been suggested to influence neutrophil function, the details remain unclear. The goal of this study was to determine the presence of the bovine FFA4 receptor and its effect on neutrophil responses. We treated bovine neutrophils with the natural and synthetic agonists of FFA4 receptor docosahexaenoic acid (DHA) and TUG-891, respectively, and assessed oxidative and no oxidative response. We detected protein and mRNA FFA4 receptor expression through immunofluorescence, immunoblot, and RT-PCR analysis. DHA and TUG-891 both increased intracellular calcium mobilisation in bovine neutrophils, with 50% effective concentrations of 99 μM and 73 μM, respectively, which was partially reduced after treatment with the FFA4 antagonist AH7614. Furthermore, DHA and TUG-891 increased matrix metalloproteinase (MMP)-9 granules release and superoxide production. AH7614 and the intracellular calcium chelator BAPTA-AM decreased the superoxide production induced by TUG-891 and by both DHA and TUG-891, respectively, suggesting a key role of intracellular calcium in FFA4 agonists-induced superoxide production. These results highlight an important mechanism of bovine neutrophil responses mediated via FFA4 receptor, which can further inform the development of new formulations for DHA-enriched feed supplements to enhance innate immunity in dairy cattle.
Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of ...replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase 𝜀 (Pol 𝜀) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol 𝜀 and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol 𝜀 trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol 𝜀 in replicative stress sensing. Together, our results demonstrate that the role of Pol 𝜀 in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.
Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression ...might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β–estradiol‐inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS‐GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β–estradiol treatment. As a proof of concept for the exploitation of this resource, β–estradiol‐induced proliferation of root hairs, dark‐induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full‐length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA‐mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl–root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.
DNA polymerase epsilon plays a key role in replicative stress sensing and signaling.
Faithful transmission of the genetic information is essential in all living organisms. DNA replication is ...therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (
Arabidopsis thaliana
). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.
PDF
