Rhamnogalacturonan lyase 1 (RGL1), as a suppressor of E3 ubiquitin ligase Arabidopsis thaliana ring zinc finger 1 (AtRZF1), is involved in dehydration response to mediate proline synthesis and pectin rhamnogalacturonan-I composition.

Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.

The role of glial and neuronal Eph/ephrin signaling in Drosophila mushroom body development and sleep and circadian behavior.

The Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species.

Secretory membrane traffic in plant-microbe interactions.

Plant defense responses include the extracellular release of defense-related molecules, such as pathogenesis-related proteins and secondary metabolites, as well as cell wall materials. This primarily depends on the trafficking of secretory vesicles to the plasma membrane, where they discharge their contents into the apoplastic space via soluble N-ethylmaleimide sensitive factor attachment protein receptor-assisted exocytosis. However, some pathogenic and symbiotic microbes have developed strategies to manipulate host plant exocytic pathways. Here, we discuss the mechanisms by which plant exocytic pathways function in immunity and how microbes have evolved to manipulate those pathways.

Synaptotagmin 4 and 5 additively contribute to Arabidopsis immunity to Pseudomonas syringae DC3000.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are essential for vesicle trafficking in plants. Vesicle-associated membrane protein 721 and 722 (VAMP721/722) are secretory vesicle-localized R-SNAREs, which are involved in a variety of biological processes in plants. Compared to VAMP721/722, a VAMP721/722-interacting plasma membrane (PM)-localized Qa-SNARE is engaged in a rather specific physiological process. This indicates that an in vivo regulator controls an interaction between a Qa-SNARE and VAMP721/722 for a specific cellular activity. We previously reported that synaptotagmin 5 (SYT5) modulates the interaction between SYP132 PM Qa-SNARE and VAMP721/722 for Arabidopsis resistance to Pseudomonas syringae DC3000. In this study, we show that defense against P. syringae DC3000 is compromised in SYT4-lacking plants, which belongs to the same subclade as SYT5. Further elevation of bacterial growth in syt4 syt5-2 plants compared to either syt4 or syt5-2 single mutant suggests that SYT4 and SYT5 play additive roles in Arabidopsis immunity to P. syringae DC3000.

Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000.

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.

Linalool Alleviates Aß42-Induced Neurodegeneration via Suppressing ROS Production and Inflammation in Fly and Rat Models of Alzheimer's Disease.

Terpenes are vital metabolites found in various plants and animals and known to be beneficial in the treatment of various diseases. Previously, our group identified terpenes that increased the survival of Alzheimer's disease (AD) model flies expressing human amyloid ß (Aß) and identified linalool as a neuroprotective terpene against Aß toxicity. Linalool is a monoterpene that is commonly present as a constituent in essential oils from aromatic plants and is known to have anti-inflammatory, anticancer, antihyperlipidemia, antibacterial, and neuroprotective properties. Although several studies have shown the beneficial effect of linalool in AD animal models, the mechanisms underlying the beneficial effect of linalool on AD are yet to be elucidated. In the present study, we showed that linalool intake increased the survival of the AD model flies during development in a dose-dependent manner, while the survival of wild-type flies was not affected even at high linalool concentrations. Linalool also decreases Aß-induced apoptosis in eye discs as well as the larval brain. Moreover, linalool intake was found to reduce neurodegeneration in the brain of adult AD model flies. However, linalool did not affect the total amount of Aß42 protein or Aß42 aggregation. Rather, linalool decreased Aß-induced ROS levels, oxidative stress, and inflammatory response in the brains of AD model flies. Furthermore, linalool attenuated the induction of oxidative stress and gliosis by Aß 1-42 treatment in the rat hippocampus. Taken together, our data suggest that linalool exerts its beneficial effects on AD by reducing Aß42-induced oxidative stress and inflammatory reactions.

SNAREs in Plant Biotic and Abiotic Stress Responses.

In eukaryotes, membraneous cellular compartmentation essentially requires vesicle trafficking for communications among distinct organelles. A donor organelle-generated vesicle releases its cargo into a target compartment by fusing two distinct vesicle and target membranes. Vesicle fusion, the final step of vesicle trafficking, is driven intrinsically by complex formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNAREs are well-conserved across eukaryotes, genomic studies revealed that plants have dramatically increased the number of SNARE genes than other eukaryotes. This increase is attributed to the sessile nature of plants, likely for more sensitive and harmonized responses to environmental stresses. In this review, we therefore try to summarize and discuss the current understanding of plant SNAREs function in responses to biotic and abiotic stresses.

CCOAOMT1, a candidate cargo secreted via VAMP721/722 secretory vesicles in Arabidopsis.

We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to understand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted (VAMP721+/-VAMP722-/- and VAMP721-/-VAMP722+/-) plants show compromised resistance to extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in both VAMP721/722-depleted lines than WT. Plants are well-known to deposit secondary cell walls as physical barriers at pathogen-attempting sites. Therefore, extracellular detection of CCOAOMT1 and impaired resistance to Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that plants secrete cell wall-modifying enzymes at least including CCOAOMT1 to reinforce the secondary cell walls for immunity.

Neuroprotective Effects of Limonene (+) against Aß42-Induced Neurotoxicity in a Drosophila Model of Alzheimer's Disease.

Forest bathing is suggested to have beneficial effects on various aspects of human health. Terpenes, isoprene based-phytochemicals emitted from trees, are largely responsible for these beneficial effects of forest bathing. Although the therapeutic effects of terpenes on various diseases have been revealed, their effects on neuronal health have not yet been studied in detail. Here, we screened 16 terpenes that are the main components of Korean forests using Drosophila Alzheimer's disease (AD) models to identify which terpenes have neuroprotective effects. Six out of the 16 terpenes, ρ-cymene, limonene (+), limonene (-), linalool, α-pinene (+), and ß-pinene (-), partially suppressed the beta amyloid 42 (Aß42)-induced rough eye phenotype when fed to Aß42-expressing flies. Among them, limonene (+) restored the decreased survival of flies expressing Aß42 in neurons during development. Limonene (+) treatment did not affect Aß42 accumulation and aggregation, but did cause to decrease cell death, reactive oxygen species levels, extracellular signal-regulated kinase phosphorylation, and inflammation in the brains or the eye imaginal discs of Aß42-expressing flies. This neuroprotective effect of limonene (+) was not associated with autophagic activity. Our results suggest that limonene (+) has a neuroprotective function against the neurotoxicity of Aß42 and, thus, is a possible therapeutic reagent for AD.

Regulation of cellular VAMP721/722 abundance in arabidopsis.

Sessile plants are continuously threatened by biotic and abiotic environmental stresses. Since stress responses are in general accompanied by growth retardation, plants in nature should tightly control timing and duration of their stress responses for sustained growth. We previously reported that vesicle-associated membrane protein (VAMP) 721 and 722 are required for growth/development and stress responses in plants. It is suggested that plants regulate expression of VAMP721/722 and/or drive VAMP721/722 to form distinct SNARE complexes with different plasma membrane (PM)-residing SNARE proteins in response to distinct stimuli. We here report that immune signaling triggered by the bacterial flg22 elicitor elevates VAMP721/722 levels in calreticulin 1 and 2 (CRT1/2)-lacking plants. Since VAMP721/722 amounts were reported not to be increased by an ER stress inducer, tunicamycin in crt1/2 plants, our results suggest that ER stress and immune signalings distinctly control cellular abundance of VAMP721/722.

Endoplasmic reticulum stress-induced accumulation of VAMP721/722 requires CALRETICULIN 1 and CALRETICULIN 2 in Arabidopsis.

Excessive demand for translation and protein folding in the endoplasmic reticulum (ER) can cause ER stress in plants. Here, we show that CALRETICULIN 1 (CRT1) and CRT2 are critical components in the accumulation of VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 during ER stress responses. We show that CRT2 interacts with VAMP722 and that CRT1/2 post-translationally maintain elevated VAMP721/722 levels under ER stress. The greater growth inhibition in VAMP721/722-deficient plants, induced by tunicamycin, suggests that plants under ER stress maintain physiological homeostasis, at least in part, by regulating VAMP721/722 levels, as VAMP721/722 are known to participate in various biological processes.

Nardostachys jatamansi Ethanol Extract Ameliorates Aß42 Cytotoxicity.

The Nardostachys jatamansi DC (NJ) root has been used as a sedative or analgesic to treat neurological symptoms and pain in traditional Korean medicine. Here, we investigate the potential effects of NJ on Alzheimer's disease (AD) and reveal the molecular mechanism through which NJ exerts its effects. The neuroprotective effect of the NJ root ethanol extract against ß amyloid (Aß) toxicity was examined in vitro using a cell culture system and in vivo using a Drosophila AD model. The NJ extract and chlorogenic acid, a major component of NJ, inhibited Aß-induced cell death in SH-SY5Y cells. Moreover, the NJ extract rescued the neurological phenotypes of the Aß42-expressing flies (decreased survival and pupariation rate and a locomotor defect) and suppressed Aß42-induced cell death in the brain. We also found that NJ extract intake reduced glial cell number, reactive oxygen species level, extracellular-signal-regulated kinase (ERK) phosphorylation, and nitric oxide level in Aß42-expressing flies, without affecting Aß accumulation. These data suggest that the neuroprotective activity of NJ might be associated with its antioxidant and anti-inflammatory properties, as well as its inhibitory action against ERK signaling; thus, NJ is a promising medicinal plant for the development of AD treatment.

Vesicle trafficking in plant immunity.

To defend against extracellular pathogens, plants primarily depend on cell-autonomous innate immunity due to the lack of the circulatory immune system including mobile immune cells. To extracellularly restrict or kill the pathogens, plant cells dump out antimicrobials. However, since antimicrobials are also toxic to plant cells themselves, they have to be safely delivered to the target sites in a separate vesicular compartment. In addition, because immune responses often requires energy otherwise used for the other metabolic processes, it is very important to properly control the duration and strength of immune responses depending on pathogen types. This can be achieved by regulating the sensing of immune signals and the delivery/discharge of extracellular immune molecules, all of which are controlled by membrane trafficking in plant cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are now considered as the minimal factors that can merge two distinct membranes of cellular compartments. Hence, in this review, known and potential immune functions of SNAREs as well as regulatory proteins will be discussed.

Phenotypic differences between Drosophila Alzheimer's disease models expressing human Aß42 in the developing eye and brain.

Drosophila melanogaster expressing amyloid-ß42 (Aß42) transgenes have been used as models to study Alzheimer's disease. Various Aß42 transgenes with different structures induce different phenotypes, which make it difficult to compare data among studies which use different transgenic lines. In this study, we compared the phenotypes of four frequently used Aß42 transgenic lines, UAS-Aß422X , UAS-Aß42BL33770 , UAS-Aß4211C39 , and UAS-Aß42H29.3 . Among the four transgenic lines, only UAS-Aß422X has two copies of the upstream activation sequence-amyloid-ß42 (UAS-Aß42) transgene, while remaining three have one copy. UAS-Aß42BL33770 has the 3' untranslated region of Drosophila α-tubulin, while the others have that of SV40. UAS-Aß4211C39 and UAS-Aß42H29.3 have the rat pre-proenkephalin signal peptide, while UAS-Aß422X and UAS-Aß42BL33770 have that of the fly argos protein. When the transgenes were expressed ectopically in the developing eyes of the flies, UAS-Aß422X transgene resulted in a strongly reduced and rough eye phenotype, while UAS-Aß42BL33770 only showed a strong rough eye phenotype; UAS-Aß42H29.3 and UAS-Aß4211C39 had mild rough eyes. The levels of cell death and reactive oxygen species (ROS) in the eye imaginal discs were consistently the highest in UAS-Aß422X , followed by UAS-Aß42BL33770 , UAS-Aß4211C39 , and UAS-Aß42H29.3 . Surprisingly, the reduction in survival during the development of these lines did not correlate with cell death or ROS levels. The flies which expressed UAS-Aß4211C39 or UAS-Aß42H29.3 experienced greatly reduced survival rates, although low levels of ROS or cell death were detected. Collectively, our results demonstrated that different Drosophila AD models show different phenotypic severity, and suggested that different transgenes may have different modes of cytotoxicity. Abbreviations: Aß42: amyloid-ß42; AD: Alzheimer's disease; UAS: upstream activation sequence.

Coriandrum sativum Suppresses Aß42-Induced ROS Increases, Glial Cell Proliferation, and ERK Activation.

Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widespread pathology that is characterized by the accumulation of amyloid [Formula: see text]-peptide (A[Formula: see text]) in the brain and various cellular abnormalities, including increased oxidative damage, an amplified inflammatory response, and altered mitogen-activated protein kinase signaling. Based on the complex etiology of AD, traditional medicinal plants with multiple effective components are alternative treatments for patients with AD. In the present study, we investigated the neuroprotective effects of an ethanol extract of Coriandrum sativum (C. sativum) leaves on A[Formula: see text] cytotoxicity and examined the molecular mechanisms underlying the beneficial effects. Although recent studies have shown the benefits of the inhalation of C. sativum oil in an animal model of AD, the detailed molecular mechanisms by which C. sativum exerts its neuroprotective effects are unclear. Here, we found that treatment with C. sativum extract increased the survival of both A[Formula: see text]-treated mammalian cells and [Formula: see text]42-expressing flies. Moreover, C. sativum extract intake suppressed [Formula: see text]-induced cell death in the larval imaginal disc and brain without affecting A[Formula: see text]42 expression and accumulation. Interestingly, the increases in reactive oxygen species levels and glial cell number in AD model flies were reduced by C. sativum extract intake. Additionally, C. sativum extract inhibited the epidermal growth factor receptor- and A[Formula: see text]-induced phosphorylation of extracellular signal-regulated kinase (ERK). The constitutively active form of ERK abolished the protective function of C. sativum extract against the [Formula: see text]-induced eye defect phenotype in Drosophila. Taken together, these results suggest that C. sativum leaves have antioxidant, anti-inflammatory, and ERK signaling inhibitory properties that are beneficial for patients with AD.

Arabidopsis immune secretory pathways to powdery mildew fungi.

Innate immune responses in host plants begin with the recognition of pathogen-specific nonself molecules and terminate with the secretion of immune molecules. In the dicotyledonous model plant, Arabidopsis thaliana, two distinct secretory pathways required for disease resistance to powdery mildew fungi have been identified so far. One is an exocytic pathway consisting of PEN1, SNAP33 and VAMP721/722 SNARE proteins, but the other is an efflux-mediated one composed of PEN2 atypical myrosinase and PEN3 ABC transporter. Based on the conservation of the mechanically same exocytic pathway in the monocotyledonous plant barely, the former is regarded as an ancient secretory pathway, whereas the latter is considered as a newly evolved one in the Brassicaceae family including Arabidopsis. We recently identified synaptotagmin 1 as an additional regulator of these two secretory pathways. With current results, we discuss how these two secretory pathways contribute to Arabidopsis immunity depending on fungal adaptedness to Arabidopsis.

Dual Effect of the Cubic Ag3PO4 Crystal on Pseudomonas syringae Growth and Plant Immunity.

We previously found that the antibacterial activity of silver phosphate crystals on Escherichia coli depends on their structure. We here show that the cubic form of silver phosphate crystal (SPC) can also be applied to inhibit the growth of a plant-pathogenic Pseudomonas syringae bacterium. SPC pretreatment resulted in reduced in planta multiplication of P. syringae. Induced expression of a plant defense marker gene PR1 by SPC alone is suggestive of its additional plant immunity-stimulating activity. Since SPC can simultaneously inhibit P. syringae growth and induce plant defense responses, it might be used as a more effective plant disease-controlling agent.

Synaptotagmin 1 Negatively Controls the Two Distinct Immune Secretory Pathways to Powdery Mildew Fungi in Arabidopsis.

PEN1, one of the plasma membrane (PM) syntaxins, comprises an immune exocytic pathway by forming the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with SNAP33 and VAMP721/722 in plants. Although this secretory pathway is also involved in plant growth and development, how plants control their exocytic activity is as yet poorly understood. Since constitutive PEN1 cycling between the PM and endocytosed vesicles is critical for its immune activity, we studied here the relationship of PEN1 to synaptotagmin 1 (SYT1) that is known to regulate endocytosis at the PM. Interestingly, syt1 plants showed enhanced disease resistance to the Arabidopsis-adapted Golovinomyces orontii fungus, and elevated protein but not transcript levels of PEN1 Calcium-dependent promotion of PEN1-SYT1 interaction suggests that SYT1 controls defense activities of the PEN1-associated secretory pathway by post-translationally modulating PEN1. Increased PEN1-SYT1 interaction and inhibited PEN1 SNARE complex induction by G. orontii additionally suggest that the adaption of phytopathogens to host plants might partly result from effective suppression of the PEN1-related secretory pathway. Further genetic analyses revealed that SYT1 also regulates the atypical peroxisomal myrosinase PEN2-associated secretory pathway.

In Vivo Screening of Traditional Medicinal Plants for Neuroprotective Activity against Aß42 Cytotoxicity by Using Drosophila Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by progressive neuronal loss with amyloid ß-peptide (Aß) plaques. Despite several drugs currently used to treat AD, their beneficial effects on AD progress remains under debate. Here, we established a rapid in vivo screening system using Drosophila AD models to assess the neuroprotective activities of medicinal plants that have been used in traditional Chinese medicine. Among 23 medicinal plants tested, the extracts from five plants, Coriandrum sativum, Nardostachys jatamansi, Polygonum multiflorum (P. multiflorum), Rehmannia glutinosa, and Sorbus commixta (S. commixta), showed protective effects against the Aß42 neurotoxicity. We further characterized the neuroprotective activity of ethanol extracts from P. multiflorum and S. commixta. Aß42-expressing flies that we used showed AD neurological phenotypes, such as decreased survival and motility and increased cell death and reactive oxygen species level. However, feeding these flies extracts from P. multiflorum or S. commixta showed strong suppression of such phenotypes. Similar results were observed in human cells, so that the treatment of P. multiflorum and S. commixta extracts increased the viability of Aß-treated SH-SY5Y cells. Moreover, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, one of the main constituents of P. multiflorum, also showed similar protective activity against Aß42 cytotoxicity in both Drosophila and human cells. Taken together, our results suggest that both P. multiflorum and S. commixta have therapeutic potential for the treatment of neurodegenerative diseases, such as AD.

Plant exocytic secretion of toxic compounds for defense.

In contrast to animals, plants do not have a circulatory system as well as mobile immune cells that allow them to protect themselves against pathogens. Instead, plants exclusively depend on the innate immune system to defend against pathogens. As typically observed in the animal innate immunity, plant immune responses are composed of pathogen detection, defense signaling which includes transcriptional reprogramming, and secretion of antimicrobial compounds. Although knowledge on recognition and subsequent signaling of pathogen-derived molecules called elicitors is now expanding, the mechanisms of how these immune molecules are excreted are yet poorly understood. Therefore, current understandings of how plants secrete defense products especially via exocytosis will be discussed in this review.