m6A-mediated upregulation of lncRNA-AC026356.1 promotes cancer stem cell maintenance in lung adenocarcinoma via activating Wnt signaling pathway.

LncRNA plays a pivotal role in the stemness and drug resistance of lung cancer. Here, we found that lncRNA-AC026356.1 was upregulated in stem spheres and chemo-resistant lung cancer cells. Our fish assay also shows that AC026356.1 was predominantly located in the cytoplasm of lung cancer cells and does not have protein-coding potential. Silencing AC026356.1 significantly inhibited proliferation and migration but increased apoptosis in A549-cisplatin (DDP) cells. Additionally, IGF2BP2 and the lncRNA-AC026356.1 positively regulated the proliferation and stemness of stem-like lung cancer cells. Further mechanistic investigation revealed that METTL14/IGF2BP2-mediated m6A modification and stabilization of the AC026356.1 RNA. Functional analysis corroborated that AC026356.1 acted as a downstream target of METTL14/IGF2BP2 and AC026356.1 silencing could block the oncogenicity of lung cancer stem-like cells. AC026356.1 expression was correlated with immune cell infiltration and T cell exhaustion. Compared with paired adjacent normal tissues, lung cancer specimens exhibited consistently upregulated METTL14/IGF2BP2/AC026356.1. M6A-modified METTL14/IGF2BP2/AC026356.1 loop may serve as a potential therapeutic target and prognostic predictor for lung cancer therapy and diagnosis in the clinic.

Silencing of the calcium-dependent protein kinase TaCDPK27 improves wheat resistance to powdery mildew.

BACKGROUND: Calcium ions (Ca2+), secondary messengers, are crucial for the signal transduction process of the interaction between plants and pathogens. Ca2+ signaling also regulates autophagy. As plant calcium signal-decoding proteins, calcium-dependent protein kinases (CDPKs) have been found to be involved in biotic and abiotic stress responses. However, information on their functions in response to powdery mildew attack in wheat crops is limited. RESULT: In the present study, the expression levels of TaCDPK27, four essential autophagy-related genes (ATGs) (TaATG5, TaATG7, TaATG8, and TaATG10), and two major metacaspase genes, namely, TaMCA1 and TaMCA9, were increased by powdery mildew (Blumeria graminis f. sp. tritici, Bgt) infection in wheat seedling leaves. Silencing TaCDPK27 improves wheat seedling resistance to powdery mildew, with fewer Bgt hyphae occurring on TaCDPK27-silenced wheat seedling leaves than on normal seedlings. In wheat seedling leaves under powdery mildew infection, silencing TaCDPK27 induced excess contents of reactive oxygen species (ROS); decreased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); and led to an increase in programmed cell death (PCD). Silencing TaCDPK27 also inhibited autophagy in wheat seedling leaves, and silencing TaATG7 also enhanced wheat seedling resistance to powdery mildew infection. TaCDPK27-mCherry and GFP-TaATG8h colocalized in wheat protoplasts. Overexpressed TaCDPK27-mCherry fusions required enhanced autophagy activity in wheat protoplast under carbon starvation. CONCLUSION: These results suggested that TaCDPK27 negatively regulates wheat resistance to PW infection, and functionally links with autophagy in wheat.

The Metacaspase TaMCA-Id Negatively Regulates Salt-Induced Programmed Cell Death and Functionally Links With Autophagy in Wheat.

Metacaspases (MCAs), a family of caspase-like proteins, are important regulators of programmed cell death (PCD) in plant defense response. Autophagy is an important regulator of PCD. This study explored the underlying mechanism of the interaction among PCD, MCAs, and autophagy and their impact on wheat response to salt stress. In this study, the wheat salt-responsive gene TaMCA-Id was identified. The open reading frame (ORF) of TaMCA-Id was 1,071 bp, coding 356 amino acids. The predicted molecular weight and isoelectric point were 38,337.03 Da and 8.45, respectively. TaMCA-Id had classic characteristics of type I MCAs domains, a typical N-terminal pro-domain rich in proline. TaMCA-Id was mainly localized in the chloroplast and exhibited nucleocytoplasmictrafficking under NaCl treatment. Increased expression of TaMCA-Id in wheat seedling roots and leaves was triggered by 150 mM NaCl treatment. Silencing of TaMCA-Id enhanced sensitivity of wheat seedlings to NaCl stress. Under NaCl stress, TaMCA-Id-silenced seedlings exhibited a reduction in activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), higher accumulation of H2O2 and O 2 . - , more serious injury to photosystem II (PSII), increase in PCD level, and autophagy activity in leaves of wheat seedlings. These results indicated that TaMCA-Id functioned in PCD through interacting with autophagy under NaCl stress, which could be used to improve the salt tolerance of crop plants.

The Calcium-Dependent Protein Kinase TaCDPK27 Positively Regulates Salt Tolerance in Wheat.

As essential calcium ion (Ca2+) sensors in plants, calcium-dependent protein kinases (CDPKs) function in regulating the environmental adaptation of plants. However, the response mechanism of CDPKs to salt stress is not well understood. In the current study, the wheat salt-responsive gene TaCDPK27 was identified. The open reading frame (ORF) of TaCDPK27 was 1875 bp, coding 624 amino acids. The predicted molecular weight and isoelectric point were 68.905 kDa and 5.6, respectively. TaCDPK27 has the closest relationship with subgroup III members of the CDPK family of rice. Increased expression of TaCDPK27 in wheat seedling roots and leaves was triggered by 150 mM NaCl treatment. TaCDPK27 was mainly located in the cytoplasm. After NaCl treatment, some of this protein was transferred to the membrane. The inhibitory effect of TaCDPK27 silencing on the growth of wheat seedlings was slight. After exposure to 150 mM NaCl for 6 days, the NaCl stress tolerance of TaCDPK27-silenced wheat seedlings was reduced, with shorter lengths of both roots and leaves compared with those of the control seedlings. Moreover, silencing of TaCDPK27 further promoted the generation of reactive oxygen species (ROS); reduced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); aggravated the injury to photosystem II (PS II); and increased programmed cell death (PCD) in wheat leaves under NaCl treatment, confirming that the TaCDPK27-silenced seedlings exhibited more NaCl injury than control seedlings. Taken together, the decrease in NaCl tolerance in TaCDPK27-silenced seedlings was due to excessive ROS accumulation and subsequent aggravation of the NaCl-induced PCD. TaCDPK27 may be essential for positively regulating salt tolerance in wheat seedlings.

Autophagic degradation of the chloroplastic 2-phosphoglycolate phosphatase TaPGLP1 in wheat.

KEY MESSAGE: TaPGLP1, a chloroplast stromal 2-phosphoglycolate phosphatase of wheat, is an ATG8-interacting protein and undergoes autophagic degradation in starvation-treated wheat mesophyll protoplasts. Selective autophagy in plants has been shown to target diverse cellular cargoes including whole chloroplasts (Chlorophagy) and several chloroplast components (Piecemeal chlorophagy). Most cargoes of selective autophagy are captured by the autophagic machinery through their direct or indirect interactions with the autophagy-essential factor ATG8. Here, we reported a new ATG8-interacting cargo of piecemeal chlorophagy, the wheat photorespiratory 2-phosphoglycolate phosphatase TaPGLP1. The TaPGLP1-mCherry fusions expressed in wheat protoplasts located in the chloroplast stroma. Strikingly, these fusions are translocated into newly formed chloroplast surface protrusions after a long time incubation of protoplasts in a nutrition-free solution. Visualization of co-expressed TaPGLP1-mCherry and the autophagy marker GFP-TaATG8a revealed physical associations of TaPGLP1-mCherry-accumulating chloroplast protrusions with autophagic structures, implying the delivery of TaPGLP1-mCherry fusions from chloroplasts to the autophagic machinery. TaPGLP1-mCherry fusions were also detected in the GFP-TaATG8a-labelled autophagic bodies undergoing degradation in the vacuoles, which suggested the autophagic degradation of TaPGLP1. This autophagic degradation of TaPGLP1 was further demonstrated by the enhanced stability of TaPGLP1-mCherry in protoplasts with impaired autophagy. Expression of TaPGLP1-mCherry in protoplasts stimulated an enhanced autophagy level probably adopted by cells to degrade the over-produced TaPGLP1-mCherry fusions. Results from gene silencing assays showed the requirement of ATG2s and ATG7s in the autophagic degradation of TaPGLP1. Additionally, TaPGLP1 was shown to interact with ATG8 family members. Collectively, our data suggest that autophagy mediates the degradation of the chloroplast stromal protein TaPGLP1 in starvation-treated mesophyll protoplasts.

Comparative transcriptomic and metabolic profiling provides insight into the mechanism by which the autophagy inhibitor 3-MA enhances salt stress sensitivity in wheat seedlings.

BACKGROUND: Salt stress hinders plant growth and production around the world. Autophagy induced by salt stress helps plants improve their adaptability to salt stress. However, the underlying mechanism behind this adaptability remains unclear. To obtain deeper insight into this phenomenon, combined metabolomics and transcriptomics analyses were used to explore the coexpression of differentially expressed-metabolite (DEM) and gene (DEG) between control and salt-stressed wheat roots and leaves in the presence or absence of the added autophagy inhibitor 3-methyladenine (3-MA). RESULTS: The results indicated that 3-MA addition inhibited autophagy, increased ROS accumulation, damaged photosynthesis apparatus and impaired the tolerance of wheat seedlings to NaCl stress. A total of 14,759 DEGs and 554 DEMs in roots and leaves of wheat seedlings were induced by salt stress. DEGs were predominantly enriched in cellular amino acid catabolic process, response to external biotic stimulus, regulation of the response to salt stress, reactive oxygen species (ROS) biosynthetic process, regulation of response to osmotic stress, ect. The DEMs were mostly associated with amino acid metabolism, carbohydrate metabolism, phenylalanine metabolism, carbapenem biosynthesis, and pantothenate and CoA biosynthesis. Further analysis identified some critical genes (gene involved in the oxidative stress response, gene encoding transcription factor (TF) and gene involved in the synthesis of metabolite such as alanine, asparagine, aspartate, glutamate, glutamine, 4-aminobutyric acid, abscisic acid, jasmonic acid, ect.) that potentially participated in a complex regulatory network in the wheat response to NaCl stress. The expression of the upregulated DEGs and DEMs were higher, and the expression of the down-regulated DEGs and DEMs was lower in 3-MA-treated plants under NaCl treatment. CONCLUSION: 3-MA enhanced the salt stress sensitivity of wheat seedlings by inhibiting the activity of the roots and leaves, inhibiting autophagy in the roots and leaves, increasing the content of both H2O2 and O2•-, damaged photosynthesis apparatus and changing the transcriptome and metabolome of salt-stressed wheat seedlings.

Silencing of ATG2 and ATG7 promotes programmed cell death in wheat via inhibition of autophagy under salt stress.

Salt stress, as an abiotic stress, limits crops production worldwide. Autophagy and programmed cell death (PCD) have been functionally linked to plant adaptation to abiotic stress. However, the relation of autophagy and PCD is still under debate and the mechanism behind remains not fully understood. In this study, salt-tolerant wheat cultivar Jimai22 was used as the experimental material, and 150 mM NaCl was added to the hydroponic culture to test the effect of salt treatment. The results showed that NaCl stress enhances autophagic activity and induced occurrence of PCD in roots and leaves of wheat seedlings. Then, the barley stripe mosaic virus-induced silencing (BSMV-VIGS) method was used to inhibit autophagy by silencing the expression of ATG2 or ATG7. The results showed that silencing of ATG2 or ATG7 significantly inhibited autophagy and impaired the tolerance of wheat to NaCl stress. Moreover, silencing of ATG2 or ATG7 disrupted the absorption of Na, Cl, K and Ca elements and led to subsequent disequilibrium of Na+, Cl-, K+ and Ca2+, induced generation of excess reactive oxygen species (ROS), decreased the antioxidant activity, damaged photosynthesis apparatus, increased the level of PCD and led to differential expression of the genes, two metacaspase genes, cysteine-rich receptor-like kinase (CRK) 10, and CRK26 in leaves of wheat seedlings under NaCl stress. The effect of the inhibitor 3-methyladenine (3-MA) on roots and leaves of wheat seedlings was in accordance with that of ATG2 and ATG7 silencing. Our results suggest that autophagy negatively regulates salt-induced PCD, or limits the scale of salt-induced PCD to avoid severe tissue death in wheat seedlings.

Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity.

BACKGROUND: Autophagy-related ATG6 proteins are pleiotropic proteins functioning in autophagy and the phosphatidylinositol 3-phosphate-signaling pathways. Arabidopsis ATG6 regulates normal plant growth, pollen development and germination, and plant responses to biotic/abiotic stresses. However, the ATG6 functions in wheat (Triticum aestivum L.), an important food crop, are lacking. RESULTS: We identified three members, TaATG6a-6c, of the ATG6 family from common wheat. TaATG6a, 6b and 6c were localized on homeologous chromosomes 3DL, 3BL and 3AL, respectively, of the allo-hexaploid wheat genome, and evidence was provided for their essential role in autophagy. The TaATG6a-GFP fusion protein was found in punctate pre-autophagosomal structures. The expression of each TaATG6 gene restored the accumulation of autophagic bodies in atg6-mutant yeast. Additionally, TaATG6 knockdown plants showed impaired constitutive and pathogen-induced autophagy and growth abnormalities under normal conditions. We also examined the expression patterns of wheat ATG6s for clues to their physiological roles, and found that their expression was induced by the fungus Blumeria graminis f. sp. tritici (Bgt), the causal agent of powdery mildew, and by abiotic stress factors. A role for TaATG6s in wheat immunity to powdery mildew was further implied when knockdowns of TaATG6s weakly compromised the broad-spectrum powdery mildew resistance gene Pm21-triggered resistance response and, conversely and significantly, enhanced the basal resistance of susceptible plants. In addition, leaf cell death was sometimes induced by growth-retarded small Bgt mycelia on susceptible TaATG6 knockdown plants after a long period of interaction. Thus, we provide an important extension of the previous characterization of plant ATG6 genes in wheat, and observed a role for autophagy genes in wheat immune responses to fungal pathogens. CONCLUSIONS: Three wheat ATG6s were identified and shown to be essential for autophagy biogenesis. Wheat ATG6s are implicated in immunity to powdery mildew, playing a weak, positive role in the Pm21-triggered resistance response and a negative role in the basal resistance of susceptible plants.

Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses.

KEY MESSAGE: The genes coding for wheat ATG4 and ATG8 were cloned and their roles in autophagy were verified. Implications of ATG4/ATG8 in wheat responses to stresses were suggested by expression profiling. Autophagy-related proteins ATG4 and ATG8 are crucial for autophagy biogenesis. ATG4 processes ATG8 precursor to expose its C-terminal glycine for phosphatidyl ethanolamine (PE) lipidation. ATG8, in the form of ATG8-PE adduct, functions in the organization dynamics of autophagic membranes. Here, we report the identification of two/nine members of the ATG4/ATG8 family from common wheat (Triticum aestivum L.). Expression of each wheat ATG4/ATG8 could complement the autophagy activity of yeast atg4/atg8 mutant cells. GFP fusion proteins of ATG8s, especially of ATG8s with innate C-terminal-exposed glycines, localized to punctate autophagic membranes. Both of purified ATG4s could cleave ATG8s in vitro, but they had different activities and different preferences for ATG8 substrates. Two times of transcript accumulation, an early one and a late one, of ATG4s/ATG8s were detected in the early phases of the Pm21- and Pm3f-triggered wheat incompatible reactions to the powdery mildew causal fungus Blumeria graminis f. sp. tritici (Bgt), and fluorescence microscopy also revealed a Bgt-induced enhanced wheat autophagy level in the Pm21-triggered incompatible reaction. Only one time of Bgt-induced transcript accumulation of ATG4s/ATG8s, corresponding to but much higher than the late one in incompatible reactions, was detected in a susceptible line isogenic to the Pm21 resistance line. These results suggested positive roles of ATG4/ATG8-associated autophagy process in the early stage and possible negative roles in the late stage of wheat immunity response to Bgt. In addition, expression of wheat ATG4s/ATG8s was also found to be upregulated by abiotic stress factors and distinctively regulated by different phytohormones.

[Advances in mechanism of Escherichia coli carbon catabolite repression].

Bacteria often sequentially utilize coexisting carbohydrates in environment and firstly select the one (frequently glucose) easiest to metabolize. This phenomenon is known as carbon catabolite repression (CCR). In existing Chinese teaching materials of molecular biology and related courses, unclear or even wrong interpretations are given about CCR mechanism. A large number of studies have shown that rather than the existence of intracellular glucose, CCR is mainly caused by the glucose transport process coupling with glucose phosphorylation via the phosphoenolpyruvate: carbohydrate phosphotransferase system PTS. The transport process leads to accumulation of dephosphorylated form of EAGlc.This form of EAGlc can bind the membrane-localized LacY protein to block the uptake of lactose inducer. cAMP functions in activation of key genes involved in PTS system to strengthen the role of inducer exclusion. In addition, dephosphorylated form of EBGlc and Yee bind global transcription repressor Mlc to ensure the expression of key genes involved in the PTS system. This review summarizes the current advancement in mechanism of Escherichia coli carbon catabolite repression.

Plastidial glutathione reductase from Haynaldia villosa is an enhancer of powdery mildew resistance in wheat (Triticum aestivum).

A full-length cDNA (Hv-GR) whose transcript accumulation increased in response to infection by Blumeria graminis DC.f.sp. tritici (Bgt) was isolated from Haynaldia villosa. Southern analysis revealed a single copy of Hv-GR present in H. villosa. This gene encodes a glutathione reductase (GR) with high similarity to chloroplastic GRs from other plant species. Chloroplastic localization of Hv-GR was confirmed by targeting of the green fluorescent protein (GFP)-Hv-GR fusion protein to chloroplasts of epidermal guard cells. Following inoculation with Bgt, transcript accumulation of Hv-GR increased in a resistant line of wheat, but no significant change was observed in a susceptible line. In vivo function of Hv-GR in converting oxidized glutathione (GSSG) to the reduced form (GSH) was verified through heterologous expression of Hv-GR in a yeast GR-deficient mutant. As expected, overexpression of this gene resulted in increased resistance of the mutant to H(2)O(2), indicating a critical role for Hv-GR in protecting cells against oxidative stress. Moreover, overexpression of Hv-GR in a susceptible wheat variety, Triticum aestivum cv. Yangmai 158, enhanced resistance to powdery mildew and induced transcript accumulation of other pathogenesis-related genes, PR-1a and PR-5, through increasing the foliar GSH/GSSG ratio. Therefore, we concluded that a high ratio of GSH to GSSG is required for wheat defense against Bgt, and that chloroplastic GR enzymes might serve as a redox mediator for NPR1 activation.

[Plant transient expression system in functional genomics].

With the development of structural and functional genomics, nowadays specific plant genome and transcriptome sequences can be cloned much easier and faster. Next step is to identify the functions of different genes and regulating elements to unravel the genetic mechanisms behind plant growth and development. Expression and its regulation are the language and dynamic property of genetic material, so expression and regulation analysis of target genes and sequences in plant cell is the basis for function study. Besides stable genetic transformation, plant transient expression system gains broad application in recent years, and its combination with other new technologies as gene shuffling, VIGS and RNAi plays a more and more important role in plant functional genomics.

[Transformation of powdery mildew resistance-related genes of wheat].

Anthocyanin synthesis regulation gene C1-Lc was used as the reporter gene to optimize the parameters of gene-gun transformation protocol through counting of red spots on wheat calli after transient expression. Wheat Beclin1 like gene TaTBL and thiosulfate sulfutransferase gene TaTST proved to have an increased expression level after induction of wheat powdery mildew fungus (Erysiphe graminis f.sp. tritici Em. Marchal.). These two resistance-related genes were constructed into expression vectors driven by the strong ubi promoter and used to perform genetic transformation on wheat cv Yangmai158 immature embryo-derived calli through particle bombardment. After two rounds of herbicide bialaphos selection and regeneration, herbicide-resistance plants were obtained, which were subsequently subjected to PCR analysis. Five TaTBL transgenic plants and six TaTST transgenic plants were identified. Pathogen inoculation of detached leaves showed that the introduction of exogenous gene increased wheat resistance level by delaying the development of powdery mildew symptoms.

Cloning and expression of peroxisomal ascorbate peroxidase gene from wheat.

A full-length cDNA encoding wheat peroxisomal ascorbate peroxidase (pAPX) was cloned by Suppression Subtractive Hybridization (SSH) and in silico approach. The cDNA was 1027 bp in length and contained a complete ORF of 876 bp, which encodes a protein of 292 amino acid residues. Its deduced amino acids sequence had 84% identity with that of pAPX from barley. The gene was designated as Ta-pAPX. The Ta-pAPX homologous genes were mapped on wheat chromosome 7A and 7D using Chinese Spring nulli-tetrasomic lines analysis. Northern analysis indicated that, after inoculation by Erysiphe graminis Dc.f.sp. tritici, the expression of Ta-pAPX gene in Yangmai5 was enhanced, but its expression in wheat-Haynaldia villosa 6VS/6AL translocation lines changed a little. The results implied that Ta-pAPX may be related to susceptibility of wheat to powdery mildew. The complete coding sequence of Ta-pAPX was cloned into an expression vector pET32 (a+) and a protein with the same deduced molecular weight (MW) was expressed in E. coli BL21 (DE3), which showed ascorbate peroxidase activity.

[Analysis of the functions of wheat resistance-related genes by a transient expression system].

Transient expression system was used to analyze the functions of three resistance- related genes: TaTBL, TaPK1 and TaTST. Target genes were constructed into plant expression vectors and transformed into leaf epidermal cells of a powdery mildew-susceptible wheat variety by gene gun. GUS gene was co-transformed with target gene to mark the transformed cells. After transformation, leaf surface was inoculated with powdery mildew conidiospores. Forty eight hours after inoculation, penetration of the fungus and formation of haustoria in transformed cells were observed to evaluate the effects of the target gene's products on the invasion of powdery mildew. The results implied that all these three genes, when transiently expressed in leaf epidermal cells of susceptible wheat variety, could partly inhibit the penetration of conidiospores and formation of haustoria, and to some extent increase the resistance of cells to powdery mildew.