Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses
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ABSTRACT: Aerenchyma is a specialized tissue consisting of longitudinal gas spaces, which enables internal movement of gases (e.g., O2, CO2, ethylene and methane), in plant roots, petioles and stems. Especially, internal transport of oxygen via aerenchyma from shoots to roots is very important for adaptation or survival of plants under waterlogged condition. To identify aerenchyma formation-associated genes expressed in maize root, we used LM combined with a microarray for monitoring genes expressed in root cortical cells under three conditions: under aerobic condition and under waterlogged condition with and without pretreatment with 1-MCP, an inhibitor of ethylene perception.
Project description:Aerenchyma is a specialized tissue consisting of longitudinal gas spaces, which enables internal movement of gases (e.g., O2, CO2, ethylene and methane), in plant roots, petioles and stems. Especially, internal transport of oxygen via aerenchyma from shoots to roots is very important for adaptation or survival of plants under waterlogged condition. To identify aerenchyma formation-associated genes expressed in maize root, we used LM combined with a microarray for monitoring genes expressed in root cortical cells under three conditions: under aerobic condition and under waterlogged condition with and without pretreatment with 1-MCP, an inhibitor of ethylene perception. For the waterlogging treatments, the primary root (but not the shoots) was waterlogged. Two and half day-old-seedlings were pre-treated with an inhibitor of ethylene perception 1-methylcyclopropene (1-MCP; 1 ppm) for 12 hours before the waterlogging treatment. Three-day-old seedlings were growing under aerated condition at the same time with other treatments as a control. Total RNA was extracted from root cortex cells from the segment of the primary root, 0.5 cm long: from 1.5 to 2 cm from the junction shoot-root derived from 3-days-old maize seedlings, and subjected to 44k oligo-DNA microarray (1. Aerated vs Hypoxia, 2. Hypoxia+MCP vs Hypoxia) with 3 biological replicates and color swaps.
Project description:To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. Aerencyma is formedonly in cortex in maise root. Therevore, aerenchyma is one of the model of programmed cell death. However, the molecular mechanism of aerenchyma formation is not understood. Therefore we isolated only cortex cells in maize primary root using laser microdissection. And microarray analysis was perforemed to identify the arechyma formation related genes. For microarray analysis, we designed 4 different experiments. Basal part and apical part of root were used for 1st experiment because arenchyma was formed in basal part of root, but not in apex. In second experiment, basal part of root with 6 hours waterlogged treatment and without treatment were used. And, as aerenchyma was induced by ethylene, ethylene and 1-MCP (inhibitor of ethylen perception) were used for experiment3 and 4. Gene expression analysis in 4 kinds of samples (cortex in basal reagion of maize primary root which were treated with waterlogged condition, aerobic condition, ethylene and 1-MCP and cortex in apical reagion of maize primary root which were treated with waterlogged conditio)
Project description:Aerenchyma is continuous gas space between shoot and roots that contributes to the internal aeration in plants. In response to excess water stress and the plant hormone ethylene, maize (Zea mays) forms aerenchyma in root cortical cells by programmed cell death (PCD). The aim of this study was to understand the molecular mechanism of ethylene-induced aerenchyma formation by identifying genes that are up- or down-regulated by ethylene treatment in the maize root cortical cells isolated by laser microdissection.
Project description:Aerenchyma is continuous gas space between shoot and roots that contributes to the internal aeration in plants. In response to excess water stress and the plant hormone ethylene, maize (Zea mays) forms aerenchyma in root cortical cells by programmed cell death (PCD). The aim of this study was to understand the molecular mechanism of ethylene-induced aerenchyma formation by identifying genes that are up- or down-regulated by ethylene treatment in the maize root cortical cells isolated by laser microdissection. Gene expression analysis in cortical cells of maize primary root
Project description:To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. Aerencyma is formedonly in cortex in maise root. Therevore, aerenchyma is one of the model of programmed cell death. However, the molecular mechanism of aerenchyma formation is not understood. Therefore we isolated only cortex cells in maize primary root using laser microdissection. And microarray analysis was perforemed to identify the arechyma formation related genes. For microarray analysis, we designed 4 different experiments. Basal part and apical part of root were used for 1st experiment because arenchyma was formed in basal part of root, but not in apex. In second experiment, basal part of root with 6 hours waterlogged treatment and without treatment were used. And, as aerenchyma was induced by ethylene, ethylene and 1-MCP (inhibitor of ethylen perception) were used for experiment3 and 4.
Project description:Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long- distance oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin depos- ited in cell walls are thought to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcrip- tion factor genes containing WRKY, AP2, and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a major constituent of the ROL barrier in roots of rice.
Project description:Aerenchyma is crucial for plant adaptation to stresses like hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades are known to regulate aerenchyma formation under hypoxic conditions, the precise mechanisms are still unclear. Moreover, cellular dynamics underlying the process of aerenchyma formation are still not well understood. We investigated the stage-dependent structural dynamics of aerenchyma in Spirodela polyrhiza (S. polyrhiza), a fast-growing aquatic herb with well-developed aerenchyma in its floating fronds. Using X-ray micro-computed tomography and histological analysis, we found that the spatial framework of aerenchyma was established before frond volume increased, driven by cell division and expansion. Additionally, the substomatal cavity connecting aerenchyma to stomata is formed via programmed cell death (PCD) closely associated with guard cell development. Transcriptome analysis and pharmacological studies further revealed that the organization of aerenchyma in S. polyrhiza is determined by the interplay between PCD and proliferation. This balance was governed by the spatiotemporal regulation of phytohormone signaling, including ethylene, abscisic acid, and salicylic acid. Our study sheds light on the structural dynamics and hormonal regulation of aerenchyma development in duckweeds, improving understanding of plant responses to environmental stresses and providing potential avenues for enhancing crop resilience under climate change.
Project description:Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long- distance oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin depos- ited in cell walls are thought to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcrip- tion factor genes containing WRKY, AP2, and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a major constituent of the ROL barrier in roots of rice. 23-d-old plants were either continued in aerated solution or transplanted into N2-flushed or stagnant deoxygenated solution for 9 h. After treating the roots of plants in aerated, stagnant, or N2-flushed conditions for 9h, the basal parts (12.5 -22.5mm below the root - shoot junction) of the adventitious roots were collected. Cells in OPR (including exodermis and sclerenchyma) were isolated using laser microdissection. RNA extracted from the isolated OPR was analysed with a 44k rice oligo-DNA microarray. Total RNAs were labeled with a Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturerM-bM-^@M-^Ys instructions. Aliquots of Cy5-labeled and Cy3-labeled cRNA (10 ng each) were used for hybridization in a rice 44K oligo-DNA microarray.
Project description:We report that CBP20 phosphorylation can regulate root growth in ethylene. We examined the small RNA expression in roots and shoots of wild type (Col) and cbp20 mutant (in Col background). Ethylene is one of the most essential hormones for plant developmental processes and stress responses. EIN2 is a key factor in ethylene signaling pathway and its function is regulated by phosphorylation. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of CBP20 is regulated by ethylene, and the phosphorylation is involved in root elongation. The constitutive phosphorylation format of CBP20 rescues the cbp20 root ethylene hyposensitive phenotype, while the constitutive de-phosphorylation format of CBP20 is unable to rescue the root phenotype of cbp20 in response to ethylene. Genome wide study on ethylene regulated gene expression and microRNA expression in the roots and shoots of both Col and cbp20, together with the result of genetics validation suggest that ethylene induced phosphorylation of CBP20 is involved in root growth and one pathway is through the regulation of microRNAs and their target genes in roots.
Project description:We report that CBP20 phosphorylation can regulate root growth in ethylene. We examined the gene expression in roots and shoots of wild type (Col) and cbp20 mutant (in Col background). Ethylene is one of the most essential hormones for plant developmental processes and stress responses. EIN2 is a key factor in ethylene signaling pathway and its function is regulated by phosphorylation. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of CBP20 is regulated by ethylene, and the phosphorylation is involved in root elongation. The constitutive phosphorylation format of CBP20 rescues the cbp20 root ethylene hyposensitive phenotype, while the constitutive de-phosphorylation format of CBP20 is unable to rescue the root phenotype of cbp20 in response to ethylene. Genome wide study on ethylene regulated gene expression and microRNA expression in the roots and shoots of both Col and cbp20, together with the result of genetics validation suggest that ethylene induced phosphorylation of CBP20 is involved in root growth and one pathway is through the regulation of microRNAs and their target genes in roots.