Cell Death Phenotype Research Articles

Spermidine mediates acetylhypusination of RIPK1 to suppress diabetes onset and progression.

It has been established that N-acetyltransferase (murine NAT1 (mNAT1) and human NAT2 (hNAT2)) mediates insulin sensitivity in type 2 diabetes. Here we show that mNAT1 deficiency leads to a decrease in cellular spermidine-a natural polyamine exhibiting health-protective and anti-ageing effects-but understanding of its mechanism is limited. We identify that mNAT1 and hNAT2 modulate a type of post-translational modification involving acetylated spermidine, which we name acetylhypusination, on receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-a key regulator of inflammation and cell death. Spermidine supplementation decreases RIPK1-mediated cell death and diabetic phenotypes induced by NAT1 deficiency in vivo. Furthermore, insulin resistance and diabetic kidney disease mediated by vascular pathology in NAT1-deficient mice can be blocked by inhibiting RIPK1. Finally, we demonstrate a decrease in spermidine and activation of RIPK1 in the vascular tissues of human patients with diabetes. Our study suggests a role for vascular pathology in diabetes onset and progression and identifies the inhibition of RIPK1 kinase as a potential therapeutic approach for the treatment of type 2 diabetes.

Microscopic Characterization of the Infectious Process, ROS Production, and Fungi Cellular Death of Alternaria alternata on Tangerine Resistant to QoIs.

Quinone outside inhibitor (QoI) fungicide resistance in Alternaria alternata populations was reported in Brazil for the first time in 2019, in São Paulo orchards, and the mutation G143A in cytochrome b (cytb) was found in resistant isolates. Our study investigated the infectious process, production of reactive oxygen species (ROS), and fungal cell death in resistant (QoI-R) and sensitive (QoI-S) A. alternata pathotype tangerine (Aapt) isolates. Morphological characterization of Aapt isolates was performed using confocal laser scanning microscopy (CLSM). Alternaria brown spot (ABS) symptoms were produced by Aapt isolates on tangelo cv. BRS Piemonte. Germination of QoI-R conidia and production of germ tubes on tangelo leaflets treated with 100 μg mL-1 of pyraclostrobin 18 h after inoculation (hai) was observed using scanning electron microscopy (SEM). At the same time, QoI-S conidial germination was inhibited on tangelo leaflets treated with pyraclostrobin. ROS production and cell death in Aapt isolates at high fungicide concentrations were observed using CLSM. QoI-S conidia exhibited high ROS production, indicating high oxidative stress. When dyed with propidium iodate (PI), QoI-S conidia emitted red fluorescence, showing cell death and confirming their sensitive phenotype. In contrast, QoI-R conidia neither produced ROS nor exhibited red fluorescence, indicating no cell death and confirming their resistant phenotype. Therefore, our findings evidence that microscopic techniques may help characterize events during fungi-plant interactions, ROS production, cell death, and Aapt phenotypes resistant and sensitive to QoIs using fluorometric protocols.

Enhancing Selectivity and Inhibitory Effects of Chemotherapy Drugs Against Myelogenous Leukemia Cells with Lippia alba Essential Oil Enriched in Citral.

Acute myelogenous leukemia (AML) is one of the most lethal cancers, lacking a definitive curative therapy due to essential constraints related to the toxicity and efficacy of conventional treatments. This study explores the co-adjuvant potential of Lippia alba essential oils (EO) for enhancing the effectiveness and selectivity of two chemotherapy agents (cytarabine and clofarabine) against AML cells. EO derived from L. alba citral chemotype were produced using optimized and standardized environmental and extraction protocols. Rational fractionation techniques were employed to yield bioactive terpene-enriched fractions, guided by relative chemical composition and cytotoxic analysis. Pharmacological interactions were established between these fractions and cytarabine and clofarabine. The study comprehensively evaluated the cytotoxic, genotoxic, oxidative stress, and cell death phenotypes induced by therapies across AML (DA-3ER/GM/EVI1+) cells. The fraction rich in citral (F2) exhibited synergistic pharmacological interactions with the studied chemotherapies, intensifying their selective cytotoxic, genotoxic, and pro-oxidant effects. This shift favored transitioning from necrosis to a programmed cell death phenotype (apoptotic). The F2-clofarabine combination demonstrated remarkable synergistic anti-leukemic performance while preserving cell integrity in healthy cells. The observed selective antiproliferative effects may be attributed to the potential dual prooxidant/antioxidant behavior of citral in L. alba EO.

PRPF40A induces inclusion of exons in GC-rich regions important for human myeloid cell differentiation.

We characterized the regulatory mechanisms and role in human myeloid cell survival and differentiation of PRPF40A, a splicing factor lacking a canonical RNA Binding Domain. Upon PRPF40A knockdown, HL-60 cells displayed increased cell death, decreased proliferation and slight differentiation phenotype with upregulation of immune activation genes. Suggestive of both redundant and specific functions, cell death but not proliferation was rescued by overexpression of its paralog PRPF40B. Transcriptomic analysis revealed the predominant role of PRPF40A as an activator of cassette exon inclusion of functionally relevant splicing events. Mechanistically, the exons exclusively upregulated by PRPF40A are flanked by short and GC-rich introns which tend to localize to nuclear speckles in the nucleus center. These PRPF40A regulatory features are shared with other splicing regulators such as SRRM2, SON, PCBP1/2, and to a lesser extent TRA2B and SRSF2, as a part of a functional network that regulates splicing partly via co-localization in the nucleus.

Chloride salt enhances plant resistance to biotic stresses.

Biotic stresses caused by bacterial and fungal pathogens damage crops; identifying treatments that enhance disease resistance provides important information for understanding plant defenses and sustainable agriculture. Salt stress affects crop yields worldwide; however, studies have focused on the toxic sodium ion, leaving the effects of the chloride ion unclear. In this study, we found that irrigation with a combination of chloride salts (MgCl2, CaCl2, and KCl) suppressed the cell death phenotype of the ceramide kinase mutant acd5. Chloride salt pre-irrigation also significantly limited the cell death caused by Pseudomonas syringae pv maculicola infection and inhibited the multiplication of this bacterial pathogen in a mechanism partially dependent on the salicylic acid pathway. Moreover, chloride salt pre-irrigation improved plant defenses against the fungal pathogen challenge, confining the lesion area caused by Botrytis cinerea infection. Furthermore, the growth of herbivorous larvae of Spodoptera exigua was retarded by feeding on chloride salt irrigated plants. Thus, our data suggest that treatment with Cl- increases broad spectrum resistance to biotic challenges.

LncRNA TubAR complexes with TUBB4A and TUBA1A to promote microtubule assembly and maintain myelination

A long-standing hypothesis proposes that certain RNA(s) must exhibit structural roles in microtubule assembly. Here, we identify a long noncoding RNA (TubAR) that is highly expressed in cerebellum and forms RNA–protein complex with TUBB4A and TUBA1A, two tubulins clinically linked to cerebellar and myelination defects. TubAR knockdown in mouse cerebellum causes loss of oligodendrocytes and Purkinje cells, demyelination, and decreased locomotor activity. Biochemically, we establish the roles of TubAR in promoting TUBB4A–TUBA1A heterodimer formation and microtubule assembly. Intriguingly, different from the hypomyelination-causing mutations, the non-hypomyelination-causing mutation TUBB4A-R2G confers gain-of-function for an RNA-independent interaction with TUBA1A. Experimental use of R2G/A mutations restores TUBB4A–TUBA1A heterodimer formation, and rescues the neuronal cell death phenotype caused by TubAR knockdown. Together, we uncover TubAR as the long-elusive structural RNA for microtubule assembly and demonstrate how TubAR mediates microtubule assembly specifically from αβ-tubulin heterodimers, which is crucial for maintenance of cerebellar myelination and activity.

Designer TALEs enable discovery of cell death-inducer genes.

Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.

Aneuploidy is Linked to Neurological Phenotypes Through Oxidative Stress

Aneuploidy, having an aberrant genome, is gaining increasing attention in neurodegenerative diseases. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. A growing body of research from numerous laboratories suggests that many neurodegenerative disorders, especially Alzheimer’s disease and frontotemporal dementia, are characterised by neuronal aneuploidy and the ensuing apoptosis, which may contribute to neuronal loss. Using Drosophila as a model, we investigated the effect of induced aneuploidy in GABAergic neurons. We found an increased proportion of aneuploidy due to Mad2 depletion in the third-instar larval brain and increased cell death. Depletion of Mad2 in GABAergic neurons also gave a defective climbing and seizure phenotype. Feeding animals an antioxidant rescued the climbing and seizure phenotype. These findings suggest that increased aneuploidy leads to higher oxidative stress in GABAergic neurons which causes cell death, climbing defects, and seizure phenotype. Antioxidant feeding represents a potential therapy to reduce the aneuploidy-driven neurological phenotype.

The chloroplast singlet oxygen-triggered biosynthesis of salicylic acid and jasmonic acid is mediated by EX1 and GUN1 in Arabidopsis.

Reactive oxygen species (ROS) and defence hormones like salicylic acid (SA) and jasmonic acid (JA) play pivotal roles in triggering cell death. However, the precise mechanism governing the interaction between ROS and SA/JA remains elusive. Recently, our research revealed that RNAi mutants with suppressed expression of PROGRAMMED CELL DEATH8 (PCD8) exhibit an overabundance of tetrapyrrole intermediates, particularly uroporphyrinogen III (Uro III), leading to the accumulation of singlet oxygen (1O2) during the transition from darkness to light, thereby instigating leaf necrosis. In this investigation, we uncovered that 1O2 stimulates biosynthesis of SA and JA, activating SA/JA signalling and the expression of responsive genes in PCD8 RNAi (pcd8) mutants. Introducing NahG or knocking out PAD4 or NPR1 significantly alleviates the cell death phenotype of pcd8 mutants, while coi1 partially mitigates the pcd8 phenotype. Further exploration revealed that EX1 and GUN1 can partially rescue the pcd8 phenotype by reducing the levels of Uro III and 1O2. Notably, mutations in EX1 mutations but not GUN1, substantially diminish SA content in pcd8 mutants compared to the wild type, implying that EX1 acts as the primary mediator of 1O2 signalling-mediated SA biosynthesis. Moreover, the triple ex1 gun1 pcd8 displays a phenotype similar to ex1. Overall, our findings underscore that the 1O2-induced cell death phenotype requires EX1/GUN1-mediated retrograde signalling in pcd8 mutants, providing novel insights into the interplay between ROS and SA/JA.

Abstract LB292: Glutathione supports lipid abundance in vivo

Abstract Glutathione (GSH), the most abundant antioxidant, scavenges free radicals and prevents the detrimental effects of oxidative stress. As a result, GSH levels are associated with protection from stress-related conditions, such as aging and cancer. Given this rationale, antioxidants are routinely consumed by the public, mainly because they are viewed as “cure-alls” and almost uniformly beneficial to a range of diseases. This is an oversimplification, as the relationship between antioxidants and diseases is much more complex. Recent evidence has implicated GSH in protecting cancer cells from oxidative stress and promoting their survival. Therefore, understanding GSH metabolism in vivo and its intricate crosstalk with other cellular pathways is vital to improving the efficacy of therapies for diseases, including cancer. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance via the LXR/SREBP signaling pathway, as well as by repressing NRF2, a transcription factor induced by oxidative stress. Overall, these findings suggest an essential function for GSH in maintaining circulating lipids levels and lipid depots, possibly as a mechanism to prevent lipid peroxidation and redox imbalances. Notably, the accumulation of oxidized lipids propagates oxidative stress and associated cell death phenotypes (i.e., ferroptosis). This also highlights a potentially novel mechanism by which GSH regulates lipid production and suggests a possible link between oxidative stress (caused by GSH deficiency) and cachexia (commonly characterized by loss of fat and lean mass), a disorder linked to cancer but poorly understood. Consequently, by elucidating the contributions of GSH to lipid homeostasis, we can better understand the basic biology surrounding GSH and obtain critical insight into potentially improving the treatment of cancer-related cachexia. Citation Format: Gloria Asantewaa, Emily T. Tuttle, Nathan P. Ward, Yun P. Kang, Yumi Kim, Madeline Kavanagh, Aaron R. Huber, Joshua Munger, Benjamin Cravatt, Calvin L. Cole, Gina M. DeNicola, Isaac S. Harris. Glutathione supports lipid abundance in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB292.

Biological Activities of Crude Extracts of Some Plant Leaves Cultivated in Vinh Long, Vietnam

This study carried on assessing the in vitro bioactivities of leaf extracts from five plants collected in Vinh Long Province, Vietnam, including extraction yield, antioxidant, antibacterial, and anticancer activities. Five crude leaf extracts were obtained by maceration with ethanol. The antioxidant, antibacterial, and anticancer activities of five extracts were evaluated separately. The results showed that the leaf extraction yields of Vitex negundo, Acrostichum aureum L., Vitex rotundifolia, Bouea macrophylla Griff, and the highest Jasminum subtriplinerve, respectively, were 1.32%, 1.4%, 1.54%, 1.72%, and 2.95%. The leaf extract of B. macrophylla Griff (BLE) had the highest antioxidant capacity, with an IC50 of 6.4 ± 0.08 μg/ml. Except for A. aureum L. and J. subtriplinerve, the other leaf extracts exhibited antibacterial properties against two Gram-positive bacteria and two Gram-negative bacteria. Notably, BLE demonstrated the maximum antibacterial activity against Listeria monocytogenes and Pseudomonas aeruginosa, with inhibition zones ranging from 15 to 16 mm and a MIC of 12.5 µg/ml. Accordingly, BLE was chosen to test for anticancer potential because its activities seemed to be greater than those of the other extracts. Especially, BLE suppressed the proliferation of HepG2 liver cancer cells with an IC50 of 72.33 ± 0.68 μg/ml. In addition, the phenotype of BLE-stimulated HepG2 cell death was typical of apoptotic cell death by analyzing the morphology and DNA fragmentation assay. Overall, the results indicated that five leaf extracts, particularly BLE, were efficient against all tested bacteria and effectively depressed the viability of HepG2 cancer cells. Furthermore, this study’s findings provide foundations for future research into the mechanisms behind these antibacterial and anticancer capabilities, as well as the knowledge of specific treatment pathways.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Abstract 1802: Melanoma addiction to GCDH

Abstract Rewiring of metabolic pathways often underlies the malignant state, including melanoma. In our previous studies we have demonstrated melanoma addiction to Gutaryl Co-dehydrogenase (GCDH), an enzyme in the lysine catabolism pathway. Our studies revealed that blocking GCDH activity in melanoma, but not colon, lung or breast cancer cells, led to cell death which abolished growth both in culture and in vivo. Important cellular component which was found to mediate melanoma addiction to GCDH was the transcription factor NRF2, which exhibited tumor suppressor function upon GCDH inhibition. Notably, coinciding with NRF2 tumor suppressor role seen upon GCDH inhibition was its glutarylation, a post translational modification which acquired NRF2 stability and ability to induce transcription of ATF4, ATF3 and CHOP which led to extensive cell death program. Knockdown of NRF2, ATF3 or DTHDK-1 effectively blocked the cell death phenotype seen upon GCDH knockdown. Analyses of patient data confirmed that low GCDH expression coincided with prolonged survival of melanoma but not colon, breast or prostate cancer patients. These findings led us to perform an unbiased screen to identify cellular components, which underlie melanoma addiction to GCDH. The results of this analysis will be discussed. Citation Format: Namratha Nadig, Sachin Verma, Ze'ev A. Ronai. Melanoma addiction to GCDH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1802.

Comprehensive analysis of MAPK gene family in upland cotton (Gossypium hirsutum) and functional characterization of GhMPK31 in regulating defense response to insect infestation

Key messageThe transcriptomic, phenotypic and metabolomic analysis of transgenic plants overexpressing GhMPK31 in upland cotton revealed the regulation of H2O2 burst and the synthesis of defensive metabolites by GhMPK31.Mitogen-activated protein kinases (MAPKs) are a crucial class of protein kinases, which play an essential role in various biological processes in plants. Upland cotton (G. hirsutum) is the most widely cultivated cotton species with high economic value. To gain a better understanding of the role of the MAPK gene family, we conducted a comprehensive analysis of the MAPK gene family in cotton. In this study, a total of 55 GhMPK genes were identified from the whole genome of G. hirsutum. Through an investigation of the expression patterns under diverse stress conditions, we discovered that the majority of GhMPK family members demonstrated robust responses to abiotic stress, pathogen stress and pest stress. Furthermore, the overexpression of GhMPK31 in cotton leaves led to a hypersensitive response (HR)-like cell death phenotype and impaired the defense capability of cotton against herbivorous insects. Transcriptome and metabolomics data analysis showed that overexpression of GhMPK31 enhanced the expression of H2O2-related genes and reduced the accumulation of defensive related metabolites. The direct evidence of GhMPK31 interacting with GhRBOHB (H2O2-generating protein) were found by Y2H, BiFC, and LCI. Therefore, we propose that the increase of H2O2 content caused by overexpression of GhMPK31 resulted in HR-like cell death in cotton leaves while reducing the accumulation of defensive metabolites, ultimately leading to a decrease in the defense ability of cotton against herbivorous insects. This study provides valuable insights into the function of MAPK genes in plant resistance to herbivorous insects.

Loss of CHCHD2 Stability Coordinates with C1QBP/CHCHD2/CHCHD10 Complex Impairment to Mediate PD-Linked Mitochondrial Dysfunction.

Novel CHCHD2 mutations causing C-terminal truncation and interrupted CHCHD2 protein stability in Parkinson's disease (PD) patients were previously found. However, there is limited understanding of the underlying mechanism and impact of subsequent CHCHD2 loss-of-function on PD pathogenesis. The current study further identified the crucial motif (aa125-133) responsible for diminished CHCHD2 expression and the molecular interplay within the C1QBP/CHCHD2/CHCHD10 complex to regulate mitochondrial functions. Specifically, CHCHD2 deficiency led to decreased neural cell viability and mitochondrial structural and functional impairments, paralleling the upregulation of autophagy under cellular stresses. Meanwhile, as a binding partner of CHCHD2, C1QBP was found to regulate the stability of CHCHD2 and CHCHD10 proteins to maintain the integrity of the C1QBP/CHCHD2/CHCHD10 complex. Moreover, C1QBP-silenced neural cells displayed severe cell death phenotype along with mitochondrial damage that initiated a significant mitophagy process. Taken together, the evidence obtained from our in vitro and in vivo studies emphasized the critical role of CHCHD2 in regulating mitochondria functions via coordination among CHCHD2, CHCHD10, and C1QBP, suggesting the potential mechanism by which CHCHD2 function loss takes part in the progression of neurodegenerative diseases.

Toxicity Tolerance in the Carcinogenesis of Environmental Cadmium.

Cadmium (Cd) is an environmental toxicant of worldwide public health significance. Diet is the main non-workplace Cd exposure source other than passive and active smoking. The intestinal absorption of Cd involves transporters for essential metals, notably iron and zinc. These transporters determine the Cd body burden because only a minuscule amount of Cd can be excreted each day. The International Agency for Research on Cancer listed Cd as a human lung carcinogen, but the current evidence suggests that the effects of Cd on cancer risk extend beyond the lung. A two-year bioassay demonstrated that Cd caused neoplasms in multiple tissues of mice. Also, several non-tumorigenic human cells transformed to malignant cells when they were exposed to a sublethal dose of Cd for a prolonged time. Cd does not directly damage DNA, but it influences gene expression through interactions with essential metals and various proteins. The present review highlights the epidemiological studies that connect an enhanced risk of various neoplastic diseases to chronic exposure to environmental Cd. Special emphasis is given to the impact of body iron stores on the absorption of Cd, and its implications for breast cancer prevention in highly susceptible groups of women. Resistance to cell death and other cancer phenotypes acquired during Cd-induced cancer cell transformation, under in vitro conditions, are briefly discussed. The potential role for the ZnT1 efflux transporter in the cellular acquisition of tolerance to Cd cytotoxicity is highlighted.

A natural single‐nucleotide polymorphism in the CAMTA3 transcription factor regulates its function and transcription of its target genes

AbstractCAMTA3, a Ca2+/calmodulin‐binding transcription factor, is a key regulator of plant immunity in Arabidopsis. Here, we identified a novel naturally occurring single‐nucleotide polymorphism that results in a missense nonconservative mutation (CAMTA3H386D) in many Arabidopsis ecotypes. This region of CAMTA3 is not part of any previously characterized regulatory domains. To study the consequence of this change on the function of CAMTA3, we introduced the CAMTA3H386D into camta3, a loss‐of‐function mutant that exhibits a constitutive cell death phenotype, chlorotic lesions on leaves, and reduced plant size. Phenotypic and molecular analysis of these lines indicated that the expression of CAMTA3H386D in the camta3 mutant did not complement the mutant phenotypes. Also, the ecotypes containing the CAMTA3H386D exhibited camta3 phenotypes. Marker genes associated with salicylic acid biosynthesis and pathogen response were upregulated in the CAMTA3H386D lines and the Arabidopsis accessions 7127 (Est‐1) and 9941 (Fei‐0), as in camta3, indicating that H386D mutation alters CAMTA3 activity in regulating the expression of known target genes. In Nicotiana benthamiana transient expression assays, CAMTA3H386D failed to induce the expression of a luciferase reporter gene driven by the rapid stress‐responsive elements (RSRE) that contain the known binding sites of CAMTA3, suggesting that CAMTA3H386D mutation impairs its ability to activate its target genes. Transgenic lines and tested natural accessions expressing CAMTA3H386D showed enhanced levels of H2O2 and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Collectively, our results indicate that the H386D mutation in a previously unknown regulatory region of CAMTA3 is essential for its function.

Validation of the Intermolecular Disulfide Bond in Caspase-2.

Caspases are a family of proteins involved in cell death. Although several caspase members have been well characterized, caspase-2 remains enigmatic. Caspase-2 has been implicated in several phenotypes, but there has been no consensus in the field about its upstream activating signals or its downstream protein targets. In addition, the unique ability of caspase-2 to form a disulfide-bonded dimer has not been studied in depth. Herein, we investigate the disulfide bond in the context of inducible dimerization, showing that disulfide bond formation is dimerization dependent. We also explore and review several stimuli published in the caspase-2 field, test ferroptosis-inducing stimuli, and study in vivo infection models. We hypothesize that the disulfide bond will ultimately prove to be essential for the evolved function of caspase-2. Proving this will require the discovery of cell death phenotypes where caspase-2 is definitively essential.

Plasma membrane-localized hexose transporter OsSWEET1b, affects sugar metabolism and leaf senescence.

OsSWEET1b is a hexose transporter protein, which localized in cell membranes and interacting with itself to form homodimer and knockout of OsSWEET1b resulted in reduced leaves sugar content and accelerating leaf senescence. In the rice genome, the SWEET gene family contains 21 homologous members, but the role of some of them in rice growth and development is still unknown. The function of the sugar transporter OsSWEET1b protein in rice was identified in this research. Expression analysis showed that the expression levels of OsSWEET1b in leaves were higher than that in other tissues. The hexose transport experiment confirmed that OsSWEET1b has glucose and galactose transporter activity in yeast. Subcellular localization indicates that OsSWEET1b protein was targeted to the plasma membrane and BiFC analysis showed that OsSWEET1b interacts with itself to form homodimers. Functional analysis demonstrated that the ossweet1b mutant plants were have reduced the sucrose, glucose, fructose, starch and galactose contents, and induced carbon starvation-related gene expression, which might lead to carbon starvation in leaves at filling stage. The ossweet1b knockout plants showed decreased chlorophyll content and antioxidant enzyme activity, and increased ROS accumulation in leaves, leading to leaf cell death and premature senescence phenotype at filling stage. In ossweet1b mutants, the leaf senescence-related gene expression levels were increased and the abundance of photosynthesis-related proteins was decreased. Loss of OsSWEET1b were affected the starch, sucrose metabolism and carbon fixation in photosynthetic organelles pathway by RNA-seq analysis. The destruction of OsSWEET1b function will cause sugar starvation, decreased photosynthesis and leaf senescence, which leading to reduced rice yield. Collectively, our results suggest that the OsSWEET1b plays a key role in rice leaves carbohydrate metabolism and leaf senescence.

Simplified Protocol to Demonstrate Gene Expression in Nicotiana benthamiana Using an Agrobacterium-Mediated Transient Assay.

Agrobacterium-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death. Key features • The protocol simplifies the widely used Agrobacterium-mediated transient gene expression assay [1] and can be completed within one week when plants are available. • Rice XB3 gene can induce a dramatic and easily identifiable cell death phenotype in Nicotiana benthamiana. • Allows identification of cell death-inducing genes and is suitable for teaching. • Compared to the currently used methods, our protocol omits the use of agroinfiltration buffer, pH meter, temperature-controlled growth chamber, centrifuge, and spectrophotometer. Graphical overview Agrobacterium infiltration (agroinfiltration) of Nicotiana benthamiana. The photo demonstrates the method of agroinfiltration into the abaxial side of leaves using a needleless syringe.