Dataset Information

A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts.

ABSTRACT: Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged Arabidopsis mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase activity and upregulated JAZ1 expression. We also assessed expression profiles and protein stability of the basic helix-loop-helix transcription factor MYC2 in wounded protoplasts. Promoter activity, gene expression, and protein stability of MYC2 were compromised, but recovered in the early stage of wounding. In the late stage, the promoter activity and expression of MYC2 were increased, but the protein stability was not changed. According to the results of the present study, this new cell-based approach will be of use in various molecular studies on plant wounding.

SUBMITTER: Son S

PROVIDER: S-EPMC8399638 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded <i>Arabidopsis</i> Protoplasts.

Son Seungmin S Kwon Miye M Im Jong Hee JH

Plants (Basel, Switzerland) 20210725 8

Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged <i>Arabidopsis</i> mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase a ...[more]

PMID: 34451563

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Project description:BackgroundMYC2, a basic helix-loop-helix (bHLH) domain-containing transcription factor, participates in the jasmonate (JA) signaling pathway and is involved in the modulation of diverse JA functions. However, a comprehensive list of MYC2-dependent JA-responsive proteins has yet to be defined.ResultsIn this paper, we report the comparative proteomics of wild-type (WT) plants and jin1-9, a MYC2 mutant plant, in response to methyl jasmonate (MeJA) treatment. Proteins from mock/MeJA-treated jin1-9 and WT samples were extracted and separated by two-dimensional gel electrophoresis. Twenty-seven JA-mediated proteins demonstrated differential expression modulated by MYC2. We observed that MYC2 negatively regulates the accumulation of JA-dependent indolic glucosinolate-related proteins and exhibits opposite effects on the biosynthetic enzymes involved aliphatic glucosinolate pathways. In addition, proteins involved in the tricarboxylic acid cycle and a majority of the MeJA-inducible proteins that are involved in multiple protective systems against oxidative stress were reduced in jin1-9/myc2 sample compared to the WT sample. These results support a positive role for MYC2 in regulating JA-mediated carbohydrate metabolism and oxidative stress tolerance.ConclusionsWe have identified MYC2-dependent jasmonate-regulated proteins in Arabidopsis thaliana by performing two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis. The observed pattern of protein expression suggests that MYC2 has opposite effects on the biosynthetic enzymes of indolic and aliphatic glucosinolate pathways and positively regulates JA-mediated carbohydrate metabolism and oxidative stress tolerance-related proteins. Furthermore, it is very interesting to note that MYC2 plays opposite roles in the modulation of a subset of JA-regulated photosynthetic proteins during short-term and long-term JA signaling. This study will enhance our understanding of the function of MYC2 in JA signaling in Arabidopsis thaliana.

Project description:BACKGROUND: Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled. RESULTS: Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q < or = 0.05) up- (> or = 2.5 fold) and downregulated (< or = -2.5 fold), respectively. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were upregulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wall-associated components were downregulated. These gene expression changes were associated with PPV infection and symptom development. Further transcriptional profiling of protoplasts transfected with a PPV infectious clone revealed the upregulation of defence and cellular signalling genes as early as 6 hours post transfection. A cross sequence comparison analysis of genes differentially regulated by PPV-infected Arabidopsis leaves against uniEST sequences derived from PPV-infected leaves of Prunus persica, a natural host of PPV, identified orthologs related to defence, metabolism and protein synthesis. The cross comparison of genes differentially regulated by PPV infection and by the infections of other positive sense RNA viruses revealed a common set of 416 genes. These identified genes, particularly the early responsive genes, may be critical in virus infection. CONCLUSION: Gene expression changes in PPV-infected Arabidopsis are the molecular basis of stress and defence-like responses, PPV pathogenesis and symptom development. The differentially regulated genes, particularly the early responsive genes, and a common set of genes regulated by infections of PPV and other positive sense RNA viruses identified in this study are candidates suitable for further functional characterization to shed lights on molecular virus-host interactions.

Project description:Phytohormones, especially jasmonates, are known to be mediators of the plant responses to wounding and herbivore feeding. Their role in such stress responses has been largely studied locally in treated leaves. However, less is known about the induced systemic distribution of phytohormone signals upon these kinds of stresses. Here, a holistic approach was performed in order to investigate the systemic phytohormone pattern in the rosette of Arabidopsis thaliana after herbivore-related wounding. Levels of different stress-related phytohormones such as jasmonates, abscisic acid, and salicylic acid were analyzed in individual leaves. We demonstrate that the typically used sampling method, where leaves are first cut and immediately frozen, causes false-positive results since cutting already induces systemic jasmonate elevations within less than 1.6 min. Therefore, this approach is not suitable to study systemic phytohormone changes in the whole plant. By developing a new method where leaves are frozen first and subsequently cut, sampling-induced phytohormone elevations could be reduced. Using this new method, we show that jasmonic acid and its active isoleucine conjugate (jasmonoyl-isoleucine) are involved in the fast systemic wound response of Arabidopsis. A systemic induction of the jasmonates' precursor, 12-oxo-phytodienoic acid, was not observed throughout our treatments. The systemic phytohormone distribution pattern is strongly linked to the vascular connections between the leaves, providing further evidence that the vascular system is used for long distance-signaling in Arabidopsis. Besides already known vascular connections, we also demonstrate that the systemic distribution of jasmonate signals can be extended to distant leaves, which are systemically but indirectly connected via another vascularly connected leaf. This holistic approach covering almost the whole Arabidopsis rosette introduces a method to overcome false-positive results in systemic phytohormone determinations and demonstrates that wounding-induced long-distance signaling includes fast changes in jasmonate levels in systemic, non-treated leaves.

Dataset Information

A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts.

Publications

A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded <i>Arabidopsis</i> Protoplasts.

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