CYSTEINE-RICH RECEPTOR-LIKE KINASE5 (CRK5) and CRK22 regulate the response to Verticillium dahliae toxins.

Cysteine-rich receptor-like kinases (CRKs) play critical roles in responses to biotic and abiotic stresses. However, the molecular mechanisms of CRKs in plant defense responses remain unknown. Here, we demonstrated that two CRKs, CRK5 and CRK22, are involved in regulating defense responses to Verticillium dahliae toxins (Vd-toxins) in Arabidopsis (Arabidopsis thaliana). Biochemical and genetic analyses showed that CRK5 and CRK22 may act upstream of MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 to regulate the salicylic acid (SA)-signaling pathway in response to Vd-toxins. In addition, MPK3 and MPK6 interact with the transcription factor WRKY70 to modulate defense responses to Vd-toxins. WRKY70 directly binds the promoter domains of the SA-signaling-related transcription factor genes TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA2) and TGA6 to regulate their expression in response to Vd-toxins. Thus, our study reveals a mechanism by which CRK5 and CRK22 regulate SA signaling through the MPK3/6-WRKY70-TGA2/6 pathway in response to Vd-toxins.

Testing the polar auxin transport model with a selective plasma membrane H+ -ATPase inhibitor.

Auxin is unique among plant hormones in that its function requires polarized transport across plant cells. A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H+ gradient across the plasma membrane (PM) established by PM H+ -adenosine triphosphatases (ATPases). However, a classical genetic approach by mutations in PM H+ -ATPase members did not result in the ablation of polar auxin distribution, possibly due to functional redundancy in this gene family. To confirm the crucial role of PM H+ -ATPases in the polar auxin transport model, we employed a chemical genetic approach. Through a chemical screen, we identified protonstatin-1 (PS-1), a selective small-molecule inhibitor of PM H+ -ATPase activity that inhibits auxin transport. Assays with transgenic plants and yeast strains showed that the activity of PM H+ -ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport. We propose that PS-1 can be used as a tool to interrogate the function of PM H+ -ATPases. Our results support the chemiosmotic model in which PM H+ -ATPase itself plays a fundamental role in polar auxin transport.

H2Bub1 Regulates RbohD-Dependent Hydrogen Peroxide Signal Pathway in the Defense Responses to Verticillium dahliae Toxins.

Histone H2B monoubiquitination (H2Bub1) plays critical roles in regulating growth and development as well as stress responses in Arabidopsis (Arabidopsis thaliana). In this study, we used wild-type and HUB1 and HUB2 loss-of-function Arabidopsis plants to elucidate the mechanisms involved in the regulation of the plant's defense responses to Verticillium dahliae toxins (Vd-toxins). We demonstrated that HUB-mediated H2Bub1 regulates the expression of the NADPH oxidase RbohD by enhancing the enrichment of histone H3 trimethylated on Lys-4 in response to Vd-toxins. RbohD-dependent hydrogen peroxide (H2O2) signaling is a critical modulator in the defense response against Vd-toxins. Moreover, H2Bub1 also affects posttranscriptional mitogen-activated protein kinase (or MPK) signaling. H2Bub1 was required for the activation of MPK3 and MPK6. MPK3 and MPK6 are involved in regulating RbohD-mediated H2O2 production. MPK3 and MPK6 are associated with protein tyrosine phosphatases (PTPs), such as Tyr-specific phosphatase1 and mitogen-activated protein kinases phosphatase1, which negatively regulated H2O2 production. In addition, H2Bub1 is involved in regulating the expression of WRKY33 WRKY33 directly binds to RbohD promoter and functions as a transcription factor to regulate the expression of RbohD Collectively, our results indicate that H2Bub1 regulates the NADPH oxidase RbohD-dependent H2O2 production and that the PTP-MPK3/6-WRKY pathway plays an important role in the regulation of RbohD-dependent H2O2 signaling in defense responses to Vd-toxins in Arabidopsis.

E2 conjugases UBC1 and UBC2 regulate MYB42-mediated SOS pathway in response to salt stress in Arabidopsis.

Histone H2B monoubiquitination (H2Bub1) is recognized as a crucial eukaryotic regulatory mechanism that controls a range of cellular processes during both development and adaptation to environmental changes. In Arabidopsis, the E2 conjugated enzymes UBIQUITIN CARRIER PROTEINs (UBCs) -1 and -2 mediate ubiquitination of H2B. Here, we elucidated the functions of UBC1 and -2 in salt-stress responses and revealed their downstream target genes. Real-time quantitative PCR assays showed that UBC1 and -2 positively regulated the salt-induced expression of MYB42 and Mitogen-Activated Protein Kinase 4 (MPK4). Chromatin immunoprecipitation assays revealed that H2Bub1 was enriched weakly on the chromatin of MYB42 and MPK4 in the ubc1,2 mutant. We further determined that UBC1/2-mediated H2Bub1 enhanced the level of histone H3 tri-methylated on K4 (H3K4me3) in the chromatin of MYB42 and MPK4 under salt-stress conditions. MPK4 interacted with and phosphorylated MYB42. The MPK4-mediated MYB42 phosphorylation enhanced the MYB42 protein stability and transcriptional activity under salt-stress conditions. We further showed that MYB42 directly bound to the SALT OVERLY SENSITIVE 2 (SOS2) promoter and mediated the rapid induction of its expression after a salt treatment. Our results indicate that UBC1 and -2 positively regulate salt-stress responses by modulating MYB42-mediated SOS2 expression.

Histone H2B monoubiquitination regulates salt stress-induced microtubule depolymerization in Arabidopsis.

Histone H2B monoubiquitination (H2Bub1) is recognized as a regulatory mechanism that controls a range of cellular processes. We previously showed that H2Bub1 was involved in responses to biotic stress in Arabidopsis. However, the molecular regulatory mechanisms of H2Bub1 in controlling responses to abiotic stress remain limited. Here, we report that HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 played important regulatory roles in response to salt stress. Phenotypic analysis revealed that H2Bub1 mutants confer decreased tolerance to salt stress. Further analysis showed that H2Bub1 regulated the depolymerization of microtubules (MTs), the expression of PROTEIN TYROSINE PHOSPHATASE1 (PTP1) and MAP KINASE PHOSPHATASE (MKP) genes - DsPTP1, MKP1, IBR5, PHS1, and was required for the activation of mitogen-activated protein kinase3 (MAP kinase3, MPK3) and MPK6 in response to salt stress. Moreover, both tyrosine phosphorylation and the activation of MPK3 and MPK6 affected MT stability in salt stress response. Thus, the results indicate that H2Bub1 regulates salt stress-induced MT depolymerization, and the PTP-MPK3/6 signalling module is responsible for integrating signalling pathways that regulate MT stability, which is critical for plant salt stress tolerance.

Histone H2B monoubiquitination is involved in regulating the dynamics of microtubules during the defense response to Verticillium dahliae toxins in Arabidopsis.

Histone H2B monoubiquitination (H2Bub) is being recognized as a regulatory mechanism that controls a range of cellular processes in plants, but the molecular mechanisms of H2Bub that are involved in responses to biotic stress are largely unknown. In this study, we used wild-type and H2Bub loss-of-function mutations of Arabidopsis (Arabidopsis thaliana) to elucidate which of its mechanisms are involved in the regulation of the plant's defense response to Verticillium dahliae (Vd) toxins. We demonstrate that the depolymerization of the cortical microtubules (MTs) was different in the wild type and the mutants in the response to Vd toxins. The loss-of-function alleles of HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 mutations present a weaker depolymerization of the MTs, and protein tyrosine phosphorylation plays a critical role in the regulation of the dynamics of MTs. Moreover, H2Bub is a positive regulator of the gene expression of protein tyrosine phosphatases. These findings provide direct evidence for H2Bub as an important modification with regulatory roles in the defense against Vd toxins and demonstrate that H2Bub is involved in modulating the dynamics of MTs, likely through the protein tyrosine phosphatase-mediated signaling pathway.

Hydrogen peroxide modulates the dynamic microtubule cytoskeleton during the defence responses to Verticillium dahliae toxins in Arabidopsis.

The molecular mechanisms of signal transduction of plants in response to infection by Verticillium dahliae (VD) are not well understood. We previously showed that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubules in Arabidopsis. In the present study, we used the wild-type, and atrbohD and atrbohF mutants of Arabidopsis to explore the mechanisms of action of H(2)O(2) signals and the dynamic microtubule cytoskeleton in defence responses. We demonstrated that H(2)O(2) may also act as an upstream signalling molecule to regulate cortical microtubule depolymerization. The depolymerization of the cortical microtubules played a functional role in the signalling pathway to mediate the expression of defence genes. The results indicate that H(2)O(2) modulates the dynamic microtubule cytoskeleton to trigger the expression of defence genes against V. dahliae toxins (VD-toxins) in Arabidopsis.

Cortical microtubule as a sensor and target of nitric oxide signal during the defence responses to Verticillium dahliae toxins in Arabidopsis.

The molecular mechanisms of signal transduction of plants in response to Verticillium dahliae (VD) are not known. Here, we show that Arabidopsis reacts to VD-toxins with a rapid burst of nitric oxide (NO) and cortical microtubule destabilization. VD-toxins treatment triggered a disruption of cortical microtubules network. This disruption can be influenced by NO production. However, cortical microtubule disruptions were not involved in regulating the NO production. The results indicated that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubule. Cortical microtubules may act as a target of NO signal and as a sensor to mediate the activation of PR-1 gene expression. These results suggested that NO production and cortical microtubule dynamics appeared to be parts of the important signalling system and are involved in the defence mechanisms to VD-toxins in Arabidopsis.

MAP65-1 is required for the depolymerization and reorganization of cortical microtubules in the response to salt stress in Arabidopsis.

Microtubules (MTs) are highly dynamical structures that play crucial roles in plant development and in response to environmental signals and stress conditions. MT-associated proteins (MAPs) play important roles in regulating the organization of MT arrays. MAP65 is a family of plant MT-bundling proteins. Here, we determined the role of MAP65-1 in the response to salt stress. MAP65-1 is involved not only in regulating the depolymerization, but also in the following reorganization of cortical MTs in salt stress responses. In addition, the depolymerization of the cortical MTs affected the survival of seedlings during salt stress, and map65-1 mutants had enhanced salt hypersensitivity levels. MAP65-1 interacted with mitogen-activated protein kinase (MPK) 3 and 6; however, only the mpk6 mutant exhibited hypersensitivity to salt stress, and MPK6 was involved in regulating the salt stress-induced depolymerization of cortical MTs. Thus, MAP65-1 plays a critical role in the response to salt stress and is required for regulating the rapid depolymerization and reorganization of cortical MTs. MAP65-1 interacts with MPK6, not MPK3, affecting the MT's dynamic instability which is critical for plant salt-stress tolerance.

Ultrastructural changes and location of beta-1, 3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of Verticillium dahliae and salicylic acid.

Calli from two cotton cultivars susceptible and resistant to Verticillium wilt, were treated with a crude toxin of Verticillium dahliae (VD-toxin) plus salicylic acid (SA). Cells treated with VD-toxin showed distinct ultrastructural changes. Cells from the susceptible cultivar displayed damage to plasma membrane and cytoplasm. The deleterious effect on cells of the resistant cultivar, with an accumulation of electron-dense precipitate in the vacuoles, was less noticeable. Exogenous SA protected callus cells from VD-toxin. We also report the localization of beta-1,3-glucanase in callus cells with immunofluorescence Labeling. Stronger fluorescence was observed in the extracellular space in resistant than in susceptible cotton; strongest in resistant cotton after 5 days of treatment with VD-toxin plus SA. The findings reported here indicate an important role of exogenous salicylic acid in the induction of resistance to VD-toxin in cotton. Coupled with an increase in beta-1,3-glucanase, cellular integrity is maintained and damage to cell wall and plasma membrane is avoided.

NO serves as a signaling intermediate downstream of H2O2 to modulate dynamic microtubule cytoskeleton during responses to VD-toxins in Arabidopsis.

Although hydrogen peroxide (H2O2) and nitric oxide (NO) can act as an upstream signaling molecule to modulate the dynamic microtubule cytoskeleton during the defense responses to Verticillium dahliae (VD) toxins in Arabidopsis, it is not known the relationship between these two signaling molecules. Here, we show that VD-toxin-induced NO accumulation was dependent on prior H2O2 production, NO is downstream of H2O2 in the signaling process, and that H2O2 acted synergistically with NO to modulate the dynamic microtubule cytoskeleton responses to VD-toxins in Arabidopsis.

Verticillium dahliae toxins-induced nitric oxide production in Arabidopsis is major dependent on nitrate reductase.

The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells.

In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ring-shaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death.

Regeneration of the secondary vascular system in poplar as a novel system to investigate gene expression by a proteomic approach.

Wood formation is a complex process composing many biological events. To access its key developmental stages, we have established a regeneration system that can mimic the initiation and differentiation of cambium cells for Chinese white poplar. Anatomical studies showed that new cambium and xylem re-appeared in sequence within a few weeks after being debarked. This provides the opportunity to follow key stages of wood formation by sampling clonal trees at different regeneration times. We used this system in combination with a proteomic approach to analyze proteins expressed in different regeneration stages. PMFs for 244 proteins differentially displayed were obtained and queried against public databases. Putative functions of 199 of these proteins were assigned and classified. Regulatory genes for cell cycle progression, differentiation and cell fate were expressed in the formation of cambial tissue, while 27 genes involved in secondary wall formation were predominantly found in the xylem developing stage. This indicates that the change of gene expression pattern is corresponding to the progression of second vascular system regeneration when and where the key events of wood development occur. Further exploration of these interesting genes may provide insight into the molecular mechanisms of wood formation.