Cell Death Phenotype Research Articles

Allelic diversity in an NLR gene BPH9 enables rice to combat planthopper variation

Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most devastating insect pests of rice (Oryza sativa L.). Currently, 30 BPH-resistance genes have been genetically defined, most of which are clustered on specific chromosome regions. Here, we describe molecular cloning and characterization of a BPH-resistance gene, BPH9, mapped on the long arm of rice chromosome 12 (12L). BPH9 encodes a rare type of nucleotide-binding and leucine-rich repeat (NLR)-containing protein that localizes to the endomembrane system and causes a cell death phenotype. BPH9 activates salicylic acid- and jasmonic acid-signaling pathways in rice plants and confers both antixenosis and antibiosis to BPH. We further demonstrated that the eight BPH-resistance genes that are clustered on chromosome 12L, including the widely used BPH1, are allelic with each other. To honor the priority in the literature, we thus designated this locus as BPH1/9 These eight genes can be classified into four allelotypes, BPH1/9-1, -2, -7, and -9 These allelotypes confer varying levels of resistance to different biotypes of BPH. The coding region of BPH1/9 shows a high level of diversity in rice germplasm. Homologous fragments of the nucleotide-binding (NB) and leucine-rich repeat (LRR) domains exist, which might have served as a repository for generating allele diversity. Our findings reveal a rice plant strategy for modifying the genetic information to gain the upper hand in the struggle against insect herbivores. Further exploration of natural allelic variation and artificial shuffling within this gene may allow breeding to be tailored to control emerging biotypes of BPH.

Developmental Epigenetic Programming of Adult Germ Cell Death Disease: Polycomb Protein EZH2–miR-101 Pathway

The Developmental Origin of Health and Disease refers to the concept that early exposure to toxicants or nutritional imbalances during perinatal life induces changes that enhance the risk of developing noncommunicable diseases in adulthood. Patients/materials & methods: An experimental model with an adult chronic germ cell death phenotype resulting from exposure to a xenoestrogen was used. A reciprocal negative feedback loop involving decreased EZH2 protein level and increased miR-101 expression was identified. In vitro and in vivo knockdown of EZH2 induced an apoptotic process in germ cells through increased levels of apoptotic factors (BIM and BAD) and DNA repair alteration via topoisomerase 2B deregulation. The increased miR-101 levels were observed in the animal blood, meaning that miR-101 may be a part of a circulating mark of germ cell death. miR-101-EZH2 pathway deregulation could represent a novel pathophysiological epigenetic basis for adult germ cell disease with environmental and developmental origins.

Effector-Triggered Immune Response in Arabidopsis thaliana Is a Quantitative Trait.

We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors.

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Dihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells

Head and neck cancer is the sixth most common cancer worldwide. Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, exhibits a wide range of biological roles including a highly efficient and specific anti-tumor activity. Here, we aimed to examine the effect of DHA on head and neck carcinoma cells and elucidate the potential mechanisms. We used five head and neck carcinoma cell lines and two non-tumorigenic normal epithelial cell lines to achieve our goals. Cells were exposed to DHA and subjected to cellular activity assays including viability, cell cycle analysis, cell death, and angiogenic phenotype. Our results show that DHA causes cell cycle arrest which is mediated through Forkhead box protein M1 (FOXM1). We also demonstrate that DHA induces ferroptosis and apoptosis in head and neck carcinoma cells. Lastly, our results show that DHA alters the angiogenic phenotype of cancer cells by reducing the expression of angiogenic factors and the ability of cancer cells to support endothelial cell tubule formation. Our study suggests that DHA specifically causes head and neck cancer cell death through contribution from both ferroptosis and apoptosis. DHA may represent an effective strategy in head and neck cancer treatment.

SHORT-ROOT Deficiency Alleviates the Cell Death Phenotype of the Arabidopsis catalase2 Mutant under Photorespiration-Promoting Conditions.

Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses.

Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes

Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

Functional and regulatory conservation of the soybean ER stress-induced DCD/NRP-mediated cell death signaling in plants

BackgroundThe developmental and cell death domain (DCD)-containing asparagine-rich proteins (NRPs) were first identified in soybean (Glycine max) as transducers of a cell death signal derived from prolonged endoplasmic reticulum (ER) stress, osmotic stress, drought or developmentally-programmed leaf senescence via the GmNAC81/GmNAC30/GmVPE signaling module. In spite of the relevance of the DCD/NRP-mediated signaling as a versatile adaptive response to multiple stresses, mechanistic knowledge of the pathway is lacking and the extent to which this pathway may operate in the plant kingdom has not been investigated.ResultsHere, we demonstrated that the DCD/NRP-mediated signaling also propagates a stress-induced cell death signal in other plant species with features of a programmed cell death (PCD) response. In silico analysis revealed that several plant genomes harbor conserved sequences of the pathway components, which share functional analogy with their soybean counterparts. We showed that GmNRPs, GmNAC81and VPE orthologs from Arabidopsis, designated as AtNRP-1, AtNRP-2, ANAC036 and gVPE, respectively, induced cell death when transiently expressed in N. benthamiana leaves. In addition, loss of AtNRP1 and AtNRP2 function attenuated ER stress-induced cell death in Arabidopsis, which was in marked contrast with the enhanced cell death phenotype displayed by overexpressing lines as compared to Col-0. Furthermore, atnrp-1 knockout mutants displayed enhanced sensitivity to PEG-induced osmotic stress, a phenotype that could be complemented with ectopic expression of either GmNRP-A or GmNRP-B. In addition, AtNRPs, ANAC036 and gVPE were induced by osmotic and ER stress to an extent that was modulated by the ER-resident molecular chaperone binding protein (BiP) similarly as in soybean. Finally, as putative downstream components of the NRP-mediated cell death signaling, the stress induction of AtNRP2, ANAC036 and gVPE was dependent on the AtNRP1 function. BiP overexpression also conferred tolerance to water stress in Arabidopsis, most likely due to modulation of the drought-induced NRP-mediated cell death response.ConclusionOur results indicated that the NRP-mediated cell death signaling operates in the plant kingdom with conserved regulatory mechanisms and hence may be target for engineering stress tolerance and adaptation in crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0843-z) contains supplementary material, which is available to authorized users.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress.

Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

Apoptosis is known as programmed cell death. Some non-apoptotic cell death is increasingly recognized as genetically controlled, or ‘regulated’. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. Here, we surveyed the landscape of pharmacologically-accessible cell death mechanisms. Of 56 caspase-independent lethal compounds, modulatory profiling revealed ten inducing three types of regulated non-apoptotic cell death. Lead optimization of one of the ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis occurs when the lipid repair enzyme GPX4 is inhibited. We found that FIN56 promotes degradation of GPX4. We performed chemoproteomics to reveal that FIN56 also binds to and activates squalene synthase, an enzyme involved in the cholesterol synthesis, in a manner independent of GPX4 degradation. These discoveries reveal that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.

Nucleocytoplasmic trafficking is essential for BAK1‐ and BKK1‐mediated cell‐death control

BRI1-ASSOCIATED KINASE1 (BAK1) was initially identified as a co-receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1-LIKE1 (BKK1) regulate a BR-independent cell-death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid- and light-dependent cell-death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1-1). Genetic analyses indicated that cell-death symptoms in a weak double mutant, bak1-3 bkk1-1, were completely suppressed by the loss-of-function mutation in SBB1, which encodes a nucleoporin (NUP)85-like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1-located sub-complex was also able to rescue the cell-death phenotype of bak1-3 bkk1-1. In addition, a DEAD-box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell-death symptoms of bak1-3 bkk1-1. Further analyses indicated that export of poly(A)(+) RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over-expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell-death phenotype of bak1-3 bkk1-1. Mutants suppressing cell-death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1- and BKK1-mediated cell-death control.

Chloroplast Activity and 3'phosphadenosine 5'phosphate Signaling Regulate Programmed Cell Death in Arabidopsis.

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.

FGFR and PTEN signaling interact during lens development to regulate cell survival

Lens epithelial cells express many receptor tyrosine kinases (RTKs) that stimulate PI3K–AKT and RAS–RAF–MEK–ERK intracellular signaling pathways. These pathways ultimately activate the phosphorylation of key cellular transcription factors and other proteins that control proliferation, survival, metabolism, and differentiation in virtually all cells. Among RTKs in the lens, only stimulation of fibroblast growth factor receptors (FGFRs) elicits a lens epithelial cell to fiber cell differentiation response in mammals. Moreover, although the lens expresses three different Fgfr genes, the isolated removal of Fgfr2 at the lens placode stage inhibits both lens cell survival and fiber cell differentiation. Phosphatase and tensin homolog (PTEN), commonly known as a tumor suppressor, inhibits ERK and AKT activation and initiates both apoptotic pathways, and cell cycle arrest. Here, we show that the combined deletion of Fgfr2 and Pten rescues the cell death phenotype associated with Fgfr2 loss alone. Additionally, Pten removal increased AKT and ERK activation, above the levels of controls, in the presence or absence of Fgfr2. However, isolated deletion of Pten failed to stimulate ectopic fiber cell differentiation, and the combined deletion of Pten and Fgfr2 failed to restore differentiation-specific Aquaporin0 and DnaseIIβ expression in the lens fiber cells.

Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1

myo-Inositol-1-phosphate synthase (MIPS) catalyzes the limiting step of inositol biosynthesis and has crucial roles in plant growth and development. In response to stress, the transcription of MIPS1 is induced and the biosynthesis of inositol or inositol derivatives is promoted by unknown mechanisms. Here, we found that the light signaling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) regulate light-induced inositol biosynthesis and oxidative stress responses by activating the transcription of MIPS1. Disruption of FHY3 and FAR1 caused light-induced cell death after dark-light transition, precocious leaf senescence, and increased sensitivity to oxidative stress. Reduction of salicylic acid (SA) accumulation by overexpression of SALICYLIC ACID 3-HYDROXYLASE largely suppressed the cell death phenotype of fhy3 far1 mutant plants, suggesting that FHY3- and FAR1-mediated cell death is dependent on SA. Furthermore, comparative analysis of chromatin immunoprecipitation sequencing and microarray results revealed that FHY3 and FAR1 directly target both MIPS1 and MIPS2. The fhy3 far1 mutant plants showed severely decreased MIPS1/2 transcript levels and reduced inositol levels. Conversely, constitutive expression of MIPS1 partially rescued the inositol contents, caused reduced transcript levels of SA-biosynthesis genes, and prevented oxidative stress in fhy3 far1. Taken together, our results indicate that the light signaling proteins FHY3 and FAR1 directly bind the promoter of MIPS1 to activate its expression and thereby promote inositol biosynthesis to prevent light-induced oxidative stress and SA-dependent cell death.

An Allelic Series of bak1 Mutations Differentially Alter bir1 Cell Death, Immune Response, Growth, and Root Development Phenotypes in Arabidopsis thaliana.

Receptor-like kinases (RLKs) mediate cell-signaling pathways in Arabidopsis thaliana, including those controlling growth and development, immune response, and cell death. The RLK coreceptor BRI1-ASSOCIATED KINASE-1 (BAK1) partners with multiple ligand-binding RLKs and contributes to their signaling in diverse pathways. An additional RLK, BAK1-INTERACTING RECEPTOR-1 (BIR1), physically interacts with BAK1, and loss-of-function mutations in BIR1 display constitutive activation of cell death and immune response pathways and dwarfism and a reduction in lateral root number. Here we show that bir1 plants display defects in primary root growth, characterize bir1 lateral root defects, and analyze expression of BIR1 and BAK1 promoters within the root. Using an allelic series of bak1 mutations, we show that loss of BAK1 function in immune response pathways can partially suppress bir1 cell death, immune response, and lateral root phenotypes and that null bak1 alleles enhance bir1 primary root phenotypes. Based on our data, we propose a model in which BIR1 functions to regulate BAK1 participation in multiple pathways.

ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions.

Somatic embryogenesis receptor-like kinase 5 in the ecotype Landsberg erecta of Arabidopsis is a functional RD LRR-RLK in regulating brassinosteroid signaling and cell death control.

In plants, LRR-RLKs play central roles in regulating perception of extracellular signals and initiation of cellular responses under various environmental challenges. Arabidopsis SERK genes, including SERK1 to SERK5, constitute a LRR-RLK sub-family. SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1 have been well characterized to function as crucial regulators in multiple physiological processes such as brassinosteroid signaling, cell death control, pathogenesis, and pollen development. Despite extremely high sequence identity with BKK1, SERK5 is reported to have no functional overlapping with BKK1, which is previously identified to regulate BR and cell death control pathways, probably due to a natural mutation in a highly conserved RD motif in the kinase domain of SERK5 in Col-0 ecotype. Through a gene sequencing analysis in several Arabidopsis accessions, we are able to identify SERK5 in Landsberg erecta (Ler) genome encoding a LRR-RLK with an intact RD motif. Overexpression of SERK5-Ler partially suppresses the BR defective phenotypes of bri1-5 and bak1-3 bkk1-1, indicating SERK5-Ler functions as a positive regulator in BR signaling. Furthermore, the interaction between SERK5-Ler and BRI1 is confirmed by yeast two-hybrid and BiFC assays, and the genetic result showing that elevated expression of a kinase-dead form of SERK5-Ler causes a dominant-negative phenotype in bri1-5. In addition, overexpression of SERK5-Ler is capable of delaying, not completely suppressing, the cell death phenotype of bak1-3 bkk1-1. In this study, we first reveal that SERK5-Ler is a biologically functional component in mediating multiple signaling pathways.

Low zinc environment induces stress signaling, senescence and mixed cell death modalities in colon cancer cells.

Currently it is not clear what type of the final cellular response (i.e. cell death modality or senescence) is induced upon chronic intracellular zinc depletion in colon cancer cells. To address this question, isogenic colon cancer lines SW480 and SW620 exposed to low zinc environment were studied over the period of 6 weeks. Low zinc environment reduced total as well as free intracellular zinc content in both cell lines. Decreased intracellular zinc content resulted in changes in cellular proliferation, cell cycle distribution and activation of stress signaling. In addition, colonocytes with low zinc content displayed increased levels of oxidative stress, changes in mitochondrial activity but in the absence of significant DNA damage. Towards the end of treatment (4th-6th week), exposed cells started to change morphologically, and typical markers of senescence as well as cell death appeared. Of two examined colon cancer cell lines, SW480 cells proved to activate predominantly senescent phenotype, with frequent form of demise being necrosis and mixed cell death modality but not apoptosis. Conversely, SW620 cells activated mostly cell death, with relatively equal distribution of apoptosis and mixed types, while senescent phenotypes and necrosis were present only in a small fraction of cell populations. Addition of zinc at the beginning of 4th week of treatment significantly suppressed cell death phenotypes in both cell lines but had no significant effect on senescence. In conclusion, presented results demonstrate variability of responses to chronic zinc depletion in colon cancer as modeled in vitro.

Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.