Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.     

Mutation in the gene encoding 1‐aminocyclopropane‐1‐carboxylate synthase 4 (CitACS4) led to andromonoecy in watermelon

Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined in watermelon is still unknown. Here we showed that the watermelon gene 1-aminocyclopropane-1-carboxylate synthase 4(Cit ACS4), expressed specifically in carpel primordia, determines the andromonoecy in watermelon. Among four single nucleotide polymorphism(SNPs) and one InDel identified in the coding region of Cit ACS4, the C364 W mutation located in the conserved box 6 was cosegregated with andromonoecy. Enzymatic analyses showed that the C364 W mutation caused a reduced activity in Cit ACS4. We believe that the reduced Cit ACS4 activity may hamper the programmed cell death in stamen primordia, leading to the formation of hermaphroditic flowers.     

Rag1 immunodeficiency‐induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1^?/? zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity‐ and apoptosis‐related genes was increased in the rag1^?/? fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1^?/? zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1^?/? zebrafish compared to their wild‐type (wt) siblings, rag1^?/? showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence‐related genes and senescence‐associated β‐galactosidase, respectively, diminished telomere length, and abnormal self‐renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1^?/? fish, as was the deficiency of the antioxidant metabolite l ‐acetylcarnitine (ALCAR) in rag1^?/? fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT‐263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Mutation in the ace‐1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a major invasive pest that has spread throughout many countries in the Mediterranean basin and parts of Asia over the last decade. The control of T. absoluta has relied heavily on the use of chemical insecticides, a strategy that has led to the evolution of resistance. In this study, biological and molecular methods were used to determine the susceptibility of five strains of T. absoluta to the organophosphate chlorpyrifos and to investigate the molecular mechanisms underlying resistance to this class of insecticides. High levels of resistance to chlorpyrifos were observed in all five strains tested. Cloning and sequencing of the gene encoding the organophosphate target site, ace‐1, of T. absoluta revealed the presence of an alanine to serine substitution at a position that has been previously linked with organophosphate resistance across a range of different insect and mite species. The presence of this mutation at high frequency in T. absoluta populations originating from various countries further supports the suggestion that the rapid expansion of this species is, in part, mediated by the resistance of this pest to chemical insecticides.     

A transcriptome‐wide study on the microRNA‐ and the Argonaute 1‐enriched small RNA‐mediated regulatory networks involved in plant leaf senescence

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence‐associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)‐enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1‐enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1‐enriched sRNAs and the miRBase‐registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1‐enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above‐identified target genes at protein level were validated as regulated by 17 AGO1‐enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1‐enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1‐enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1‐enriched sRNAs in rice, indicating species‐specific functions of these sRNA–target pairs. These results could advance our understanding of the sRNA‐involved molecular processes modulating leaf senescence.     

The Arabidopsis CERK1‐associated kinase PBL27 connects chitin perception to MAPK activation

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola. PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo. Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.     

Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1‐family effectors relies on EDS1‐dependent immunity

Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N. benthamiana when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR‐NB‐LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium‐mediated protein expression experiments in wild‐type and EDS1‐deficient (eds1) N. benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ‐mediated cell death suppression in N. benthamiana is due to the attenuation of Agrobacterium‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

The impact of Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant

Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, a genetic‐ and molecular‐based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T‐DNA insertional mutants did not display an apparent phenotype. Crossing of the single mutants did not allow the isolation of double‐mutant plants, whereas self‐pollinating the S1?/? S2+/? sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar‐supplemented media in vitro, were shown to be AtSnAK1 AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/? S2?/?. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T‐loop of the SnRK1 catalytic subunit to be non‐phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild‐type plant growth and development is dependent on the presence of SnAK in vivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling in planta.     

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady‐state maintenance and turnover of plant LDs, particularly in non‐seed tissues, are relatively unknown. Previously, we showed that the LD‐associated proteins (LDAPs) are a family of plant‐specific, LD surface‐associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two‐hybrid library using the Arabidopsis LDAP3 isoform as ‘bait’ in an effort to identify other novel LD protein constituents. One of the candidate LDAP3‐interacting proteins was Arabidopsis At5g16550, which is a plant‐specific protein of unknown function that we termed LDIP (LDAP‐interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α‐helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T‐DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds.     

A COP1‐PIF‐HEC regulatory module fine‐tunes photomorphogenesis in Arabidopsis

Light responses mediated by the photoreceptors play crucial roles in regulating different aspects of plant growth and development. An E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)1/SUPPRESSOR OF PHYA (SPA), one of the central repressors of photomorphogenesis, is critical for maintaining skotomorphogenesis. It targets several positive regulators of photomorphogenesis for degradation in darkness. Recently, we revealed that basic helix‐loop‐helix factors, HECATEs (HECs), function as positive regulators of photomorphogenesis by directly interacting and antagonizing the activity of another group of repressors called PHYTOCHROME‐INTERACTING FACTORs (PIFs). It was also shown that HECs are partially degraded in the dark through the ubiquitin/26S proteasome pathway. However, the underlying mechanism of HEC degradation in the dark is still unclear. Here, we show that HECs also interact with both COP1 and SPA proteins in darkness, and that HEC2 is directly targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway. Moreover, COP1‐mediated polyubiquitylation and degradation of HEC2 are enhanced by PIF1. Therefore, the ubiquitylation and subsequent degradation of HECs are significantly reduced in both cop1 and pif mutants. Consistent with this, the hec mutants partially suppress photomorphogenic phenotypes of both cop1 and pifQ mutants. Collectively, our work reveals that the COP1/SPA‐mediated ubiquitylation and degradation of HECs contributes to the coordination of skoto/photomorphogenic development in plants.     

Sphingosine‐1‐phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti‐myeloperoxidase antibody‐positive IgG

Cumulating evidences suggested an important role of sphingosine‐1‐phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)‐ANCA‐positive IgG‐mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO‐ANCA‐positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane‐bound and soluble ICAM‐1 and VCAM‐1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO‐ANCA‐positive IgG‐induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO‐ANCA‐positive IgG. S1P enhanced MPO‐ANCA‐positive IgG‐induced membrane‐bound and soluble ICAM‐1/VCAM‐1 up‐regulation of GEnCs. Soluble ICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG increased upon pre‐incubation of S1PR1 antagonist, while pre‐incubation of GEnCs with the S1PR1 agonist down‐regulated sICAM‐1 level. Blocking S1PR2‐4 reduced sICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG. Pre‐incubation with S1PR5 agonist could increase sICAM‐1 level in the supernatants of GEnC stimulated by S1P and MPO‐ANCA‐positive IgG. S1P can enhance MPO‐ANCA‐positive IgG‐mediated GEnC activation through S1PR2‐5.