The miR172/TOE3 module regulates resistance to tobacco mosaic virus in tobacco.

The outcome of certain plant-virus interaction is symptom recovery, which is accompanied with the emergence of asymptomatic tissues in which the virus accumulation decreased dramatically. This phenomenon shows the potential to reveal critical molecular factors for controlling viral disease. MicroRNAs act as master regulators in plant growth, development, and immunity. However, the mechanism by which miRNA participates in regulating symptom recovery remains largely unknown. Here, we reported that miR172 was scavenged in the recovered tissue of tobacco mosaic virus (TMV)-infected Nicotiana tabacum plants. Overexpression of miR172 promoted TMV infection, whereas silencing of miR172 inhibited TMV infection. Then, TARGET OF EAT3 (TOE3), an APETALA2 transcription factor, was identified as a downstream target of miR172. Overexpression of NtTOE3 significantly improved plant resistance to TMV infection, while knockout of NtTOE3 facilitated virus infection. Furthermore, transcriptome analysis indicated that TOE3 promoted the expression of defense-related genes, such as KL1 and MLP43. Overexpression of these genes conferred resistance of plant against TMV infection. Importantly, results of dual-luciferase assay, chromatin immunoprecipitation-quantitative PCR, and electrophoretic mobility shift assay proved that TOE3 activated the transcription of KL1 and MLP43 by binding their promoters. Moreover, overexpression of rTOE3 (the miR172-resistant form of TOE3) significantly reduced TMV accumulation compared to the overexpression of TOE3 (the normal form of TOE3) in miR172 overexpressing Nicotiana benthamiana plants. Taken together, our study reveals the pivotal role of miR172/TOE3 module in regulating plant immunity and in the establishment of recovery in virus-infected tobacco plants, elucidating a regulatory mechanism integrating plant growth, development, and immune response.

NtG3BPL1 confers resistance to chilli veinal mottle virus through promoting the degradation of 6K2 in tobacco.

The RNA regulatory network is a complex and dynamic regulation in plant cells involved in mRNA modification, translation, and degradation. Ras-GAP SH3 domain-binding protein (G3BP) is a scaffold protein for the assembly of stress granules (SGs) and is considered an antiviral component in mammals. However, the function of G3BP during virus infection in plants is still largely unknown. In this study, four members of the G3BP-like proteins (NtG3BPLs) were identified in Nicotiana tabacum and the expression levels of NtG3BPL1 were upregulated during chilli veinal mottle virus (ChiVMV) infection. NtG3BPL1 was localized in the nucleus and cytoplasm, forming cytoplasmic granules under transient high-temperature treatment, whereas the abundance of cytoplasmic granules was decreased under ChiVMV infection. Overexpression of NtG3BPL1 inhibited ChiVMV infection and delayed the onset of symptoms, whereas knockout of NtG3BPL1 promoted ChiVMV infection. In addition, NtG3BPL1 directly interacted with ChiVMV 6K2 protein, whereas 6K2 protein had no effect on NtG3BPL1-derived cytoplasmic granules. Further studies revealed that the expression of NtG3BPL1 reduced the chloroplast localization of 6K2-GFP and the NtG3BPL1-6K2 interaction complex was localized in the cytoplasm. Furthermore, NtG3BPL1 promoted the degradation of 6K2 through autophagy pathway, and the accumulation of 6K2 and ChiVMV was affected by autophagy activation or inhibition in plants. Taken together, our results demonstrate that NtG3BPL1 plays a positive role in tobacco resistance against ChiVMV infection, revealing a novel mechanism of plant G3BP in antiviral strategy.

MYB30 Regulates Submergence Tolerance by Repressing Ethylene Biosynthesis via ACS7 in Arabidopsis.

Floods impose detrimental effects on natural and agro-ecosystems, leading to a significant loss of worldwide crop production. Global climate change has even worsened this situation. Flooding is a continuous process including two stages of submergence and re-oxygenation, and both are harmful to plant growth and development, resulting in a serious decline in crop yield. Therefore, the understanding of plant flooding tolerance and developing flooding-resistant crops are of great significance. Here, we report that the Arabidopsis thaliana (Arabidopsis) R2R3-MYB transcription factor MYB30 participates in plant submergence response through 1-aminocyclopropane-1-carboxylic acid synthase 7 (ACS7) by repressing ethylene (ET) biosynthesis. The MYB30 loss-of-function mutant exhibits reduced submergence tolerance with a higher level of ET production, whereas the MYB30-overexpressing plant displays enhanced submergence tolerance and repressed ET production. The coding gene of ACS7 might be a direct target of MYB30 during the submergence response. MYB30 binds to the promoter of ACS7 and represses its transcription. The ACS7 loss-of-function mutant with defect in ET biosynthesis displays enhanced submergence tolerance, whereas plants overexpressing ACS7 exhibit a submergence-sensitive phenotype. Genetic analysis shows that ACS7 functions downstream of MYB30 in both ET biosynthesis and submergence response. Taken together, our work revealed a novel transcriptional regulation that modulates submergence response in plants.

Experimental investigations on the growth of wall-attached bubble in total dissolved gas supersaturated water.

Due to dam discharge, waterfalls, sudden increases in water temperature and oxygen production by photosynthesis, the total dissolved gas (TDG) in water is often supersaturated, which may have serious effects on aquatic ecology. When the atmospheric pressure is lower than the TDG pressure in water, the supersaturated dissolved gas in water will slowly release into air. Wall-attached bubbles were formed during the TDG release process. The generation and departure of wall-attached bubbles influence the release process of TDG in water. To simulate the growth period of the wall-attached bubbles under different pressures, a decompression experimental device was designed to record the supersaturated TDG release process. Based on experimental data and mathematical calculations, the quantitative relationship between the bubble growth rate and environmental pressure was obtained. The supersaturated TDG dissipation rate increases monotonically with increasing relative vacuum degree. Applied the calculation method about the wall-attached bubble growth rate, a formula of the supersaturated TDG adsorption flux was proposed, and a prediction method of the TDG release coefficient was established. The simulation results show that with the increasing relative vacuum degree, the TDG release coefficient increases correspondingly, and the adsorption from wall surface area can be obviously promoted. This study provides an important theoretical basis for the accurate calculation of the TDG release process and provides a scientific basis for the accurate prediction of the spatial and temporal distribution of supersaturated TDG under different pressure and solid wall conditions.

NtAGO1 positively regulates the generation and viral resistance of dark green islands in Nicotiana tabacum.

Dark green islands (DGIs) are the outcome of post-transcriptional gene silencing (PTGS) in antiviral immunity, but their characteristics related to PTGS remain largely unknown. In this study, the cucumber mosaic virus (CMV) was inoculated on Nicotiana tabacum plants to explore the PTGS features of DGIs. Our results showed that higher expressions of PTGS-associated genes, especially NtAGO1, present in DGIs. To investigate the role of NtAGO1 in the generation and the antiviral effect of DGIs, NtAGO1 was then over-expressed or knocked out in N. tabacum plants through agrobacterium-mediated genetic transformation. The results showed that more DGIs with larger areas appeared on NtAGO1 over-expressed plants, accompanied by less virus accumulation, less reactive oxygen species production, and seldom membrane damage, whereas fewer DGIs appeared on NtAGO1 knockout plants with more damage on infected plants. In addition, the NtAGO1-participated antiviral process could promote the transduction of the salicylic acid-mediated defense pathway. Taken together, our results indicate that DGIs are maintained by a stronger PTGS mechanism, and NtAGO1 positively regulates the generation and viral resistance of DGIs in N. tabacum.

MYB30 and ETHYLENE INSENSITIVE3 antagonistically modulate root hair growth in Arabidopsis.

Root hair (RH) is essential for plant nutrient acquisition and the plant-environment communication. Here we report that transcription factors MYB30 and ETHYLENE INSENSITIVE3 (EIN3) modulate RH growth/elongation in Arabidopsis in an antagonistic way. The MYB30 loss-of-function mutant displays enhanced RH length, whereas the RH elongation in MYB30-overexpressing plants is highly repressed. MYB30 physically interacts with EIN3, a master transcription factor in ethylene signaling. MYB30 directly binds the promoter region of ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) and represses its transcription. RSL4 loss-of-function suppresses the enhanced RH growth in myb30 mutant plants. Ethylene enhances MYB30-EIN3 complex formation, and reduces the association between MYB30 and RSL4 promotor via the action of EIN3. MYB30 and EIN3 antagonistically regulate the expression of RSL4 and a subset of core RH genes in a genome-wide way. Taken together, our work revealed a novel transcriptional network that modulates RH growth in plants.

Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum.

Plant symptoms are derived from specific interactions between virus and host components. However, little is known about viral or host factors that participate in the establishment of systemic necrosis. Here, we showed that helper component proteinase (HCPro), encoded by Chilli veinal mottle virus (ChiVMV), could directly interact with catalase 1 (CAT1) and catalase 3 (CAT3) in the cytoplasm of tobacco (Nicotiana tabacum) plants to facilitate viral infection. In vitro, the activities of CAT1 and CAT3 were inhibited by the interaction between HCPro and CATs. The C-terminus of HCPro was essential for their interaction and was also required for the decrease of enzyme activities. Interestingly, the mRNA and protein level of CATs were up-regulated in tobacco plants in response to ChiVMV infection. Nicotiana tabacum plants with HCPro overexpression or CAT1 knockout were more susceptible to ChiVMV infection, which was similar to the case of H2O2-pre-treated plants, and the overexpression of CAT1 inhibited ChiVMV accumulation. Also, neither CAT1 nor CAT3 could affect the RNA silencing suppression (RSS) activity of HCPro. Our results showed that the interaction between HCPro and CATs promoted the development of plant systemic necrosis, revealing a novel role for HCPro in virus infection and pathogenicity.

The role of phytochromes in Nicotiana tabacum against Chilli veinal mottle virus.

It has been reported that phytochrome A (phyA) and phytochrome B (phyB) are potent regulators of plant defense. However, the mechanisms that phytochromes use to interfere with plant resistance to viral infection remain largely unclear. In this study, Chilli veinal mottle virus (ChiVMV) was used to investigate the role of phytochromes in response to biotic stress. Our results showed that the phytochromes mutant phyAphyB28 plants displayed more serious necrosis and dwarf phenotypes compared to that of wild type plants (WT) after ChiVMV infection. qRT-PCR and Western blot analyses indicated that the expression and accumulation of ChiVMV were higher in phyAphyB28 mutants than that in WT plants. The leakage (EL) and the content of thiobarbituric acid-reactive substance (TBARS) suggested that phyAphyB28 mutants suffered more severe membrane damage than that of WT plants. In addition, extensive ROS accumulated in phyAphyB28 mutants after ChiVMV infection, whereas ROS production in WT plants were much less than mutant plants. The activities of antioxidant enzymes were down-regulated in phyAphyB28 mutants when compared with that in WT plants under ChiVMV infection. Besides, the contents of endogenous SA, JA and the expression of both hormones signaling related genes were lower in phyAphyB28 mutants compared to that in WT plants. Application of exogenous SA and JA could alleviate disease symptoms. Taken together, these results demonstrated that phyA and phyB positively regulated plant defense responses to ChiVMV infection and this process was dependent on the SA and JA defense pathways.

N gene enhances resistance to Chilli veinal mottle virus and hypersensitivity to salt stress in tobacco.

Plants use multiple mechanisms to fight against pathogen infection. One of the major mechanisms involves the disease resistance (R) gene, which specifically mediates plant defense. Recent studies have shown that R genes have broad spectrum effects in response to various stresses. N gene is the resistance gene specifically resistant to Tobacco mosaic virus (TMV). However, the role of N gene in abiotic stress and other viral responses remains obscure. In this study, we investigated the mechanisms by which N regulates plant defense responses under Chilli veinal mottle virus (ChiVMV) infection and salt stress. Here, we monitored the physiological and molecular changes of tobacco plants under virus attack. The results showed that when tobaccoNN and tobacconn plants were exposed to ChiVMV, tobaccoNN plants displayed higher susceptibility at five days post infection (dpi), while tobacconn plants exhibited higher susceptibility at 20 dpi. In addition, accumulation of reactive oxygen species (ROS) and expression of HARPIN-INDUCED1(NtHIN1) were higher in tobaccoNN plants than in tobacconn plants at 5 dpi. Interestingly, the pathogenesis-related gene (NtPR1 and NtPR5), the activities of antioxidant enzymes, and the content of salicylic acid (SA) in tobaccoNN plants increased compared with those in tobacconn plants. It was suggested that the N gene induced a hypersensitive response (HR) and enhanced the systemic resistance of plants in response to ChiVMV via the SA-dependent signaling pathway. In addition, the N gene was also induced significantly by salt stress. However, tobaccoNN plants showed hypersensitivity toward increased salt stress, and this hypersensitivity was dependent on abscisic acid and jasmonic acid but not SA. Taken together, our results indicate that the N gene appears to be important in the plant response to ChiVMV infection and salt stress.

Phytochrome A and B Negatively Regulate Salt Stress Tolerance of Nicotiana tobacum via ABA-Jasmonic Acid Synergistic Cross-Talk.

Light signaling and phytohormones play important roles in plant growth, development, and biotic and abiotic stress responses. However, the roles of phytochromes and cross-talk between these two signaling pathways in response to salt stress in tobacco plants remain underexplored. Here, we explored the defense response in phytochrome-defective mutants under salt stress. We monitored the physiological and molecular changes of these mutants under salt stress conditions. The results showed that phytochrome A (phyA), phytochrome B (phyB) and phyAphyB (phyAB) mutants exhibited improved salt stress tolerance compared with wild-type (WT) plants. The mutant plants had a lower electrolyte leakage (EL) and malondialdehyde (MDA) concentration than WT plants, and the effect was clearly synergistic in the phyAB double mutant plants. Furthermore, the data showed that the transcript levels of defense-associated genes and the activities of some antioxidant enzymes in the mutant plants were much higher than those in WT plants. Additionally, the results indicated that phytochrome signaling strongly modulates the expression of endogenous abscisic acid (ABA) and jasmonic acid (JA) of Nicotiana tobacum in response to salt stress. To illustrate further the relationship between phytochrome and phytohormone, we measured the expression of defense genes and phytochrome. The results displayed that salt stress and application of methyl jasmonate (MeJA) or ABA up-regulated the transcript levels of salt response-associated genes and inhibited the expression of NtphyA and NtphyB. Foliar application of inhibitors of ABA and JA further confirmed that JA co-operated with ABA in phytochrome-mediated salt stress tolerance.

α-Expansin EXPA4 Positively Regulates Abiotic Stress Tolerance but Negatively Regulates Pathogen Resistance in Nicotiana tabacum.

Since they function as cell wall-loosening proteins, expansins can affect plant growth, developmental processes and environmental stress responses. Our previous study demonstrated that changes in Nicotiana tabacum α-expansin 4 (EXPA4) expression affect the sensitivity of tobacco to Tobacco mosaic virus [recombinant TMV encoding green fluorescent protein (TMV-GFP)] infection by Agrobacterium-mediated transient expression. In this study, to characterize the function of tobacco EXPA4 further, EXPA4 RNA interfernce (RNAi) mutants and overexpression lines were generated and assayed for their tolerance to abiotic stress and resistance to pathogens. First, the differential phenotypes and histomorphology of transgenic plants with altered EXPA4 expression indicated that EXPA4 is essential for normal tobacco growth and development. By utilizing tobacco EXPA4 mutants with abiotic stress, it was demonstrated that RNAi mutants have increased hypersensitivity to salt and drought stress. In contrast, the overexpression of EXPA4 in tobacco conferred greater tolerance to salt and drought stress, as indicated by less cell damage, higher fresh weight, higher soluble sugar and proline accumulation, and higher expression levels of several stress-responsive genes. In addition, the overexpression lines were more susceptible to the viral pathogen TMV-GFP when compared with the wild type or RNAi mutants. The induction of the antioxidant system, several defense-associated phytohormones and gene expression was down-regulated in overexpression lines but up-regulated in RNAi mutants when compared with the wild type following TMV-GFP infection. In addition, EXPA4 overexpression also accelerated the disease development of Pseudomonas syringae DC3000 on tobacco. Taken together, these results suggested that EXPA4 appears to be important in tobacco growth and responses to abiotic and biotic stress.

Mitochondrial alternative oxidase-dependent autophagy involved in ethylene-mediated drought tolerance in Solanum lycopersicum.

Mitochondrial alternative oxidase (AOX) is involved in a large number of plant physiological processes, such as growth, development and stress responses; however, the exact role of AOX in response to drought remains unclear. In our study, we provide solid evidences that the activated AOX capacity positively involved in ethylene-induced drought tolerance, in tomato (Solanum lycopersicum), accompanied by the changing level of hydrogen peroxide (H2 O2 ) and autophagy. In AOX1a-RNAi plants, the ethylene-induced drought tolerance was aggravated and associated with decreasing level of autophagy. The H2 O2 level was relatively higher in AOX1a-RNAi plants, whereas it was lower in AOX1a-overexpressing (35S-AOX1a-OE) plants after 1-(aminocarbonyl)-1-cyclopropanecarboxylic acid (ACC) pretreatment in the 14th day under drought stress. Interestingly, the accumulation of autophagosome was accompanied by the changing level of reactive oxygen species (ROS) in AOX transgenic tomato under drought stress whether or not pretreated with ACC. Pharmacological scavenging of H2 O2 accumulation in AOX1a-RNAi (aox19) stimulated autophagy acceleration under drought stress, and it seems that AOX-dependent ROS signalling is critical in triggering autophagy. Lower levels of ROS signalling positively induce autophagy activity, whereas higher ROS level would lead to rapid programmed cell death (PCD), especially in ethylene-mediated drought tolerance. Moreover, ethylene-induced autophagy during drought stress also can be through ERF5 binding to the promoters of ATG8d and ATG18h. These results demonstrated that AOX plays an essential role in ethylene-induced drought tolerance and also played important roles in mediating autophagy generation via balancing ROS level.

Alpha-momorcharin enhances Tobacco mosaic virus resistance in tobaccoNN by manipulating jasmonic acid-salicylic acid crosstalk.

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29 kDa found in plants. This protein has been shown to be effective against a broad range of human viruses and also has anti-tumor activities. However, the mechanism by which α-MMC induces plant defense responses and regulates the N gene to promote resistance to the Tobacco mosaic virus (TMV) is still not clear. By using pharmacological and infection experiments, we found that α-MMC enhances TMV resistance of tobacco plants containing the N gene (tobaccoNN). Our results showed that plants pretreated with 0.5 mg/ml α-MMC could relieve TMV-induced oxidative damage, had enhanced the expression of the N gene and increased biosynthesis of jasmonic acid (JA) and salicylic acid (SA). Moreover, transcription of JA and SA signaling pathway genes were increased, and their expression persisted for a longer period of time in plants pretreated with α-MMC compared with those pretreated with water. Importantly, exogenous application of 1-Aminobenzotriazole (ABT, SA inhibitor) and ibuprofen (JA inhibitor) reduced α-MMC induced plant resistance under viral infection. Thus, our results revealed that α-MMC enhances TMV resistance of tobaccoNN plants by manipulating JA-SA crosstalk.

OTS1-dependent deSUMOylation increases tolerance to high copper levels in Arabidopsis.

The conjugation of SUMO (small ubiquitin-like modifier) to protein substrates is a reversible process (SUMOylation/deSUMOylation) that regulates plant development and stress responses. The essential metal copper (Cu) is required for normal plant growth, but excess amounts are toxic. The SUMO E3 ligase, SIZ1, and SIZ1-mediated SUMOylation function in plant tolerance to excess Cu. It is unknown whether deSUMOylation also contributes to Cu tolerance in plants. Here, we report that OTS1, a protease that cleaves SUMO from its substrate proteins, participates in Cu tolerance in Arabidopsis thaliana (Arabidopsis). OTS1 loss-of-function mutants (ots1-2 and ots1-3) displayed increased sensitivity to excess Cu. Redox homeostasis and the balance between SUMOylation and deSUMOylation were disrupted in the ots1-3 mutant under excess Cu conditions. The ots1-3 mutant accumulated higher levels of Cu in both shoots and roots compared to wild type. Specific Cu-related metal transporter genes were upregulated due to the loss-of-function of OTS1, which might explain the high Cu levels in ots1-3. These results suggest that the SUMOylation/deSUMOylation machinery is activated in response to excess Cu, and modulates Cu homeostasis and tolerance by regulating both Cu uptake and detoxification. Together, our findings provide insight into the biological function and regulatory role of SUMOylation/deSUMOylation in plant tolerance to Cu.

Tobacco alpha-expansin EXPA4 plays a role in Nicotiana benthamiana defence against Tobacco mosaic virus.

MAIN CONCLUSION: Tobacco EXPA4 plays a role in Nicotiana benthamiana defence against virus attack and affects antioxidative metabolism and phytohormone-mediated immunity responses in tobacco. Expansins are cell wall-loosening proteins known for their endogenous functions in cell wall extensibility during plant growth. The effects of expansins on plant growth, developmental processes and environment stress responses have been well studied. However, the exploration of expansins in plant virus resistance is rarely reported. In the present study, virus-induced gene silencing (VIGS) and Agrobacterium-mediated transient overexpression were conducted to investigate the role of Nicotiana tabacum alpha-expansin 4 (EXPA4) in modulating Tobacco mosaic virus (TMV-GFP) resistance in Nicotiana benthamiana. The results indicated that silencing of EXPA4 reduced the sensitivity of N. benthamiana to TMV-GFP, and EXPA4 overexpression accelerated virus reproduction on tobacco. In addition, our data suggested that the changes of virus accumulation in response to EXPA4 expression levels could further affect the antioxidative metabolism and phytohormone-related pathways in tobacco induced by virus inoculation. EXPA4-silenced plants with TMV-GFP have enhanced antioxidant enzymes activities, which were down-regulated in virus-inoculated 35S:EXPA4 plants. Salicylic acid accumulation and SA-mediated defence genes induced by TMV-GFP were up-regulated in EXPA4-silenced plants, but depressed in 35S:EXPA4 plants. Furthermore, a VIGS approach was used in combination with exogenous phytohormone treatments, suggesting that EXPA4 has different responses to different phytohormones. Taken together, these results suggested that EXPA4 plays a role in tobacco defence against viral pathogens.

RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum.

Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various viruses that induce leaf mosaic in infected plants. The inoculation of Cucumber mosaic virus (CMV) in Nicotiana tabacum produced a commonly occurring sequence of classic patterns of DGIs and LGTs. Previous studies confirmed that there are significant differences between DGIs and LGTs in terms of physiology, biochemistry and molecular biology, but the mechanisms by which DGIs form remain unclear. To investigate the global gene expression changes that occur in these special tissues, individual differential gene expression tag libraries were constructed from three total RNA samples isolated from DGIs, LGTs and control plants (CK) and were sequenced using an Illumina HiSeqTM 2000. An analysis of differentially expressed genes (DEGs) and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. These analyses revealed the differences between DGIs, LGTs and CK. GO enrichment and KEGG pathway analyses suggested that several pathways related to photosynthesis and chlorophyll metabolism were enriched in DGIs compared to LGTs and CK. Several pathways related to apoptosis were significantly up-regulated in LGTs compared to DGIs. Additionally, we identified sets of DEGs that may be related to the formation or development of DGIs and LGTs. Our systematic analyses provide comprehensive transcriptomic information regarding DGIs and LGTs in CMV-infected N. tabacum. These data will help characterize the detailed mechanisms of DGI and LGT formation.

The Roles of Alpha-Momorcharin and Jasmonic Acid in Modulating the Response of Momordica charantia to Cucumber Mosaic Virus.

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein with a molecular weight of 29 kDa that is found in Momordica charantia, and has been shown to be effective against a broad range of human viruses as well as having anti-tumor activities. However, the role of endogenous α-MMC under viral infection and the mechanism of the anti-viral activities of α-MMC in plants are still unknown. To study the effect of α-MMC on plant viral defense and how α-MMC increases plant resistance to virus, the M. charantia-cucumber mosaic virus (CMV) interaction system was investigated. The results showed that the α-MMC level was positively correlated with the resistance of M. charantia to CMV. α-MMC treatment could alleviate photosystem damage and enhance the ratio of glutathione/glutathione disulfide in M. charantia under CMV infection. The relationship of α-MMC and defense related phytohormones, and their roles in plant defense were further investigated. α-MMC treatment led to a significant increase of jasmonic acid (JA) and vice versa, while there was no obvious relevance between salicylic acid and α-MMC. In addition, reactive oxygen species (ROS) were induced in α-MMC-pretreated plants, in a similar way to the ROS burst in JA-pretreated plants. The production of ROS in both ibuprofen (JA inhibitor) and (α-MMC+ibuprofen)-pretreated plants was reduced markedly, leading to a greater susceptibility of M. charantia to CMV. Our results indicate that the anti-viral activities of α-MMC in M. charantia may be accomplished through the JA related signaling pathway.

Ethylene and hydrogen peroxide are involved in brassinosteroid-induced salt tolerance in tomato.

Crosstalk between phytohormone pathways is essential in plant growth, development and stress responses. Brassinosteroids (BRs) and ethylene are both pivotal plant growth regulators, and the interaction between these two phytohormones in the tomato response to salt stress is still unclear. Here, we explored the mechanism by which BRs affect ethylene biosynthesis and signaling in tomato seedlings under salt stress. The activity of 1-aminocyclopropane-1-carboxylate synthase (ACS), an ethylene synthesis enzyme, and the ethylene signaling pathway were activated in plants pretreated with BRs. Scavenging of ethylene production or silencing of ethylene signaling components inhibited BR-induced salt tolerance and blocked BR-induced activities of several antioxidant enzymes. Previous studies have reported that BRs can induce plant tolerance to a variety of environmental stimuli by triggering the generation of H2O2 as a signaling molecule. We also found that H2O2 might be involved in the crosstalk between BRs and ethylene in the tomato response to salt stress. Simultaneously, BR-induced ethylene production was partially blocked by pretreated with a reactive oxygen species scavenger or synthesis inhibitor. These results strongly demonstrated that ethylene and H2O2 play important roles in BR-dependent induction of plant salt stress tolerance. Furthermore, we also investigated the relationship between BR signaling and ethylene signaling pathways in plant processes responding to salt stress.

Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering.

The phenomenon of delayed flowering after the application of nitrogen (N) fertilizer has long been known in agriculture, but the detailed molecular basis for this phenomenon is largely unclear. Here we used a modified method of suppression-subtractive hybridization to identify two key factors involved in N-regulated flowering time control in Arabidopsis thaliana, namely ferredoxin-NADP(+)-oxidoreductase and the blue-light receptor cryptochrome 1 (CRY1). The expression of both genes is induced by low N levels, and their loss-of-function mutants are insensitive to altered N concentration. Low-N conditions increase both NADPH/NADP(+) and ATP/AMP ratios, which in turn affect adenosine monophosphate-activated protein kinase (AMPK) activity. Moreover, our results show that the AMPK activity and nuclear localization are rhythmic and inversely correlated with nuclear CRY1 protein abundance. Low-N conditions increase but high-N conditions decrease the expression of several key components of the central oscillator (e.g., CCA1, LHY, and TOC1) and the flowering output genes (e.g., GI and CO). Taken together, our results suggest that N signaling functions as a modulator of nuclear CRY1 protein abundance, as well as the input signal for the central circadian clock to interfere with the normal flowering process.