Interaction between regulation of autophagy, stress responses and growth in Arabidopsis thaliana

Plants have evolved sophisticated mechanisms to balance between growth and stress tolerance upon changing environmental conditions. Autophagy is a critical process for recycling of cytoplasmic materials during nutrient remodeling and stress responses. Upon activation, the materials to be degraded are engulfed by a double-membrane vesicle called an autophagosome, which delivers the cargo to the vacuole for degradation and recycling. Studies in plants have revealed genes that are involved in the core machinery of autophagosome formation and delivery, and key regulators of autophagy. However, the upstream regulators of autophagy and the functions of autophagy in balancing growth and stress tolerance remain unclear. This dissertation summarizes my efforts in studying the regulation and functions of autophagy in Arabidopsis thaliana. Previous studies identified a key regulator of autophagy, target of rapamycin (TOR), being a negative regulator of autophagy, and a positive regulator of plant growth. TOR has been suggested to be a nutrient sensor that activates autophagy during nutrient deficiency. Here we have assessed the extent to which TOR controls autophagy activation under abiotic stress. Through overexpression of TOR and activation of TOR activity by auxin, we have revealed that not only nutrient stress, but also salt and osmotic stresses regulate autophagy through a TOR-dependent pathway. In addition, oxidative stress and ER stress-induced autophagy are independent of TOR. Our results also have shown that auxin negatively regulates autophagy through the TOR-dependent pathway, providing a new mechanism of auxin-regulated stress tolerance in plants. Another plant hormone that promotes plant growth is brassinosteroids (BRs). Our results identified a new link between the BR and TOR signaling through phosphorylation of TOR by a BR-regulated kinase Brassinazole-Insensitive 2 (BIN2). BRs were also characterized as negative regulators of autophagy, and BR regulates autophagy and plant growth through the interaction with the TOR signaling pathway. This reveals a new mechanism of balancing plant growth and stress response through interaction between hormone signaling and regulation of autophagy. We have also identified the TOR-independent pathway of autophagy regulation upon ER stress. ER stress is triggered when cells accumulate excessive unfolded and misfolded proteins, which then leads to the unfolded protein response (UPR). IRE1 is a dual-function protein kinase and ribonuclease, and one of the isoform, IRE1b, was shown to be dependent in the activation of autophagy upon ER stress. Here we have shown that the ribonuclease function of IRE1b is responsible for autophagy regulation, and we have identified three genes in the Regulated Ire1-Dependent Decay of Messenger RNA (RIDD) pathway that negatively regulate autophagy induction upon ER stress. In summary, our results reveal that autophagy induced upon abiotic stresses is regulated through TOR-dependent and –independent pathways, and the TOR signaling pathway interacts with auxin and BR hormone signaling to regulate plant growth and stress responses. ER stress regulates autophagy is dependent of IRE1b ribonuclease function

IRE1B degrades RNAs encoding proteins that interfere with the induction of autophagy by ER stress in Arabidopsis thaliana

Macroautophagy/autophagy is a conserved process in eukaryotes that contributes to cell survival in response to stress. Previously, we found that endoplasmic reticulum (ER) stress induces autophagy in plants via a pathway dependent upon AT5G24360/IRE1B (INOSITOL REQUIRING 1–1), an ER membrane-anchored factor involved in the splicing of AT1G42990/BZIP60 (basic leucine zipper protein 60) mRNA. IRE1B is a dual protein kinase and ribonuclease, and here we determined the involvement of the protein kinase catalytic domain, nucleotide binding and RNase domains of IRE1B in activating autophagy. We found that the nucleotide binding and RNase activity of IRE1B, but not its protein kinase activity or splicing target BZIP60, are required for ER stress-mediated autophagy. Upon ER stress, the RNase activity of IRE1B engages in regulated IRE1-dependent decay of messenger RNA (RIDD), in which mRNAs of secreted proteins are degraded by IRE1 upon ER stress. Twelve genes most highly targeted by RIDD were tested for their role in inhibiting ER stress-induced autophagy, and 3 of their encoded proteins, AT1G66270/BGLU21 (β-glucosidase 21), AT2G16005/ROSY1/ML (MD2-related lipid recognition protein) and AT5G01870/PR-14 (pathogenesis-related protein 14), were found to inhibit autophagy upon overexpression. From these findings, IRE1B is posited to be a ‘licensing factor’ linking ER stress to autophagy by degrading the RNA transcripts of factors that interfere with the induction of autophagy

Clinicopathological characteristics and survival in lung signet ring cell carcinoma: a population-based study

Lung signet-ring cell carcinoma (LSRCC) is a very rare type of lung cancer, the clinical characteristics, and prognosis of which remain to be clarified. In order to explore the clinicopathological and survival-related factors associated with LSRCC, we performed a large population-based cohort analysis of data included in the Surveillance, Epidemiology, and End Results (SEER) registry from 2001 to 2015. A total of 752 LSRCC and 7518 lung mucinous adenocarcinoma (LMAC) patients were incorporated into our analysis, with respective mean ages of 63.8 and 67.5 years at the time of diagnosis. LSRCC patients were significantly more likely than LMAC patients to have distant-stage disease (72.1% vs. 45.8%, p < 0.0001), tumors of a high pathological grade (40.6% vs. 10.8%, p < 0.0001), have undergone chemotherapy (62.1% vs. 39.9%, p<0.0001), be male (52.7% vs. 48.5%, p = 0.03), and be < 40 years old (3.3% vs. 1.3%, p = 0.022), whereas they were less likely to have undergone surgical treatment (52.4% vs. 77.0%, p < 0.0001). LSRCC and LMAC patients exhibited median overall survival (OS) duration of 8 and 18 months (p < 0.0001), respectively, although these differences were not significant after adjusting for confounding variables. Independent factors associated with a favorable patient prognosis included a primary site in the middle or lower lung lobe, underwent surgery, and underwent chemotherapy. However, age ≥80 years, higher grade, distant summary stage disease, and T4 stage disease were linked to poor prognosis. Patient age, tumor grade, primary tumor site, summary stage, T stage, surgery, and chemotherapy were all significantly associated with LSRCC patient prognosis

Quantitative proteomics reveals extensive lysine ubiquitination in the Arabidopsis root proteome and uncovers novel transcription factor stability states

Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function and yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich peptides containing the di-glycine (diGly) remnant of ubiquitin and coupled that with isobaric labeling to enable quantification, from up to 16-multiplexed samples, for plant tissues. Collectively, we identified 7,130 diGly-modified lysine residues sites arising from 3,178 proteins in Arabidopsis primary roots. These data include ubiquitin proteasome dependent ubiquitination events as well as ubiquitination events associated with auxin treatment. Gene Ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and root morphogenesis. We determined the ubiquitinated lysine residues that directly regulate the stability of the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) using site directed mutagenesis and in vivo degradation assays. These comprehensive site-level ubiquitinome profiles provide a wealth of data for future studies related to modulation of biological processes mediated by this posttranslational modification in plants

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

Thermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes. transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic.In this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically

Interaction between regulation of autophagy, stress responses and growth in Arabidopsis thaliana

Plants have evolved sophisticated mechanisms to balance between growth and stress tolerance upon changing environmental conditions. Autophagy is a critical process for recycling of cytoplasmic materials during nutrient remodeling and stress responses. Upon activation, the materials to be degraded are engulfed by a double-membrane vesicle called an autophagosome, which delivers the cargo to the vacuole for degradation and recycling. Studies in plants have revealed genes that are involved in the core machinery of autophagosome formation and delivery, and key regulators of autophagy. However, the upstream regulators of autophagy and the functions of autophagy in balancing growth and stress tolerance remain unclear. This dissertation summarizes my efforts in studying the regulation and functions of autophagy in Arabidopsis thaliana. Previous studies identified a key regulator of autophagy, target of rapamycin (TOR), being a negative regulator of autophagy, and a positive regulator of plant growth. TOR has been suggested to be a nutrient sensor that activates autophagy during nutrient deficiency. Here we have assessed the extent to which TOR controls autophagy activation under abiotic stress. Through overexpression of TOR and activation of TOR activity by auxin, we have revealed that not only nutrient stress, but also salt and osmotic stresses regulate autophagy through a TOR-dependent pathway. In addition, oxidative stress and ER stress-induced autophagy are independent of TOR. Our results also have shown that auxin negatively regulates autophagy through the TOR-dependent pathway, providing a new mechanism of auxin-regulated stress tolerance in plants. Another plant hormone that promotes plant growth is brassinosteroids (BRs). Our results identified a new link between the BR and TOR signaling through phosphorylation of TOR by a BR-regulated kinase Brassinazole-Insensitive 2 (BIN2). BRs were also characterized as negative regulators of autophagy, and BR regulates autophagy and plant growth through the interaction with the TOR signaling pathway. This reveals a new mechanism of balancing plant growth and stress response through interaction between hormone signaling and regulation of autophagy. We have also identified the TOR-independent pathway of autophagy regulation upon ER stress. ER stress is triggered when cells accumulate excessive unfolded and misfolded proteins, which then leads to the unfolded protein response (UPR). IRE1 is a dual-function protein kinase and ribonuclease, and one of the isoform, IRE1b, was shown to be dependent in the activation of autophagy upon ER stress. Here we have shown that the ribonuclease function of IRE1b is responsible for autophagy regulation, and we have identified three genes in the Regulated Ire1-Dependent Decay of Messenger RNA (RIDD) pathway that negatively regulate autophagy induction upon ER stress. In summary, our results reveal that autophagy induced upon abiotic stresses is regulated through TOR-dependent and –independent pathways, and the TOR signaling pathway interacts with auxin and BR hormone signaling to regulate plant growth and stress responses. ER stress regulates autophagy is dependent of IRE1b ribonuclease function.</p

Structure Characteristics and Influencing Factors of Cross-Border Electricity Trade: A Complex Network Perspective

Electricity is one of the most widely used forms of energy. However, environmental pollution from electricity generation and the mismatch between electricity supply and demand have long been bothering economies across the world. Under this background, cross-border electricity trade provides a new direction for sustainable development. Based on the complex network approach, this paper aims to explore the structural characteristics and evolution of cross-border electricity trade networks and to figure out the factors influencing the formation of the network by using the more advanced network analysis method—ERGM. The results show that: (1) The scale of the electricity trade network is expanding, but there are still many economies not involved. (2) The centrality of the network shifts from west to east. The level of internal electricity interconnection is high in Europe, and Asian countries’ coordination role in cross-border electricity trade networks is enhanced. (3) Cross-border electricity trade helps to reduce CO2 emissions, achieve renewable energy transformation, and reduce power supply and demand mismatch. Large gaps in GDP, electricity prices, industrial structure, geographical distance and institutional distance between economies are not conducive to form the cross-border trade network, while the common language is on the contrary

TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana

Autophagy is a critical process for recycling of cytoplasmic materials during environmental stress, senescence and cellular remodeling. It is upregulated under a wide range of abiotic stress conditions and is important for stress tolerance. Autophagy is repressed by the protein kinase target of rapamycin (TOR), which is activated in response to nutrients and in turn upregulates cell growth and translation and inhibits autophagy. Down-regulation of TOR in Arabidopsis thaliana leads to constitutive autophagy and to decreased growth, but the relationship to stress conditions is unclear. Here, we assess the extent to which TOR controls autophagy activation by abiotic stress. Overexpression of TOR inhibited autophagy activation by nutrient starvation, salt and osmotic stress, indicating that activation of autophagy under these conditions requires down-regulation of TOR activity. In contrast, TOR overexpression had no effect on autophagy induced by oxidative stress or ER stress, suggesting that activation of autophagy by these conditions is independent of TOR function. The plant hormone auxin has been shown previously to up-regulate TOR activity. To confirm the existence of two pathways for activation of autophagy, dependent on the stress conditions, auxin was added exogenously to activate TOR, and the effect on autophagy under different conditions was assessed. Consistent with the effect of TOR overexpression, the addition of the auxin NAA inhibited autophagy during nutrient deficiency, salt and osmotic stress, but not during oxidative or ER stress. NAA treatment was unable to block autophagy induced by a TOR inhibitor or by a mutation in the TOR complex component RAPTOR1B, indicating that auxin is upstream of TOR in the regulation of autophagy. We conclude that repression of auxin-regulated TOR activity is required for autophagy activation in response to a subset of abiotic stress conditions

TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana

Autophagy is a critical process for recycling of cytoplasmic materials during environmental stress, senescence and cellular remodeling. It is upregulated under a wide range of abiotic stress conditions and is important for stress tolerance. Autophagy is repressed by the protein kinase target of rapamycin (TOR), which is activated in response to nutrients and in turn upregulates cell growth and translation and inhibits autophagy. Down-regulation of TOR in Arabidopsis thaliana leads to constitutive autophagy and to decreased growth, but the relationship to stress conditions is unclear. Here, we assess the extent to which TOR controls autophagy activation by abiotic stress. Overexpression of TOR inhibited autophagy activation by nutrient starvation, salt and osmotic stress, indicating that activation of autophagy under these conditions requires down-regulation of TOR activity. In contrast, TOR overexpression had no effect on autophagy induced by oxidative stress or ER stress, suggesting that activation of autophagy by these conditions is independent of TOR function. The plant hormone auxin has been shown previously to up-regulate TOR activity. To confirm the existence of two pathways for activation of autophagy, dependent on the stress conditions, auxin was added exogenously to activate TOR, and the effect on autophagy under different conditions was assessed. Consistent with the effect of TOR overexpression, the addition of the auxin NAA inhibited autophagy during nutrient deficiency, salt and osmotic stress, but not during oxidative or ER stress. NAA treatment was unable to block autophagy induced by a TOR inhibitor or by a mutation in the TOR complex component RAPTOR1B, indicating that auxin is upstream of TOR in the regulation of autophagy. We conclude that repression of auxin-regulated TOR activity is required for autophagy activation in response to a subset of abiotic stress conditions.This article is published as Pu, Yunting, Xinjuan Luo, and Diane C. Bassham. "TOR-dependent and-independent pathways regulate autophagy in Arabidopsis thaliana." Frontiers in plant science 8 (2017): 1204. doi: 10.3389/fpls.2017.01204.</p