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RNA-seq of enriched barley stomatal complexes versus total leaves

ABSTRACT: We intend to identify transporters and their regulators involved in barley stomatal movement as well as components of the ABA signaling pathway. We isolated epidermal peels where only stomatal guard cells and their subsidiary cells survived, while other epidermal cell were mechanically disrupted and sequenced the resulting RNAs. Thus we analyzed transcripts differentially expressed between the barley stomatal complex and total leaves. These data served as the first overview of genes expressed in the stomatal complex and for cloning of relevant transporters.

INSTRUMENT(S): Illumina HiSeq 3000

ORGANISM(S): Hordeum vulgare

SUBMITTER: Peter Ache

PROVIDER: E-MTAB-5877 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Barley guard cell and subsidiary cell specific transcripts

Project description:We prepared RNA from total leaves and epidermal peels, containing living guard cells (GC) and subsidiary cells (SC) only. In the third sample we blended epidermal peels to mechanically destroy the SC, leading to a pure guard cells fraction. By RNAseq and substraction of GC from SC transcripts, we were able to reliably assign transcripts to the barley SCs and GCs.

2023-12-31 | E-MTAB-10534 | biostudies-arrayexpress

Microarray analysis of scap1 mutant and wild type in Arabidopsis thaliana guard cell protoplast

Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation. We isolated guard cell protoplasts from 4-week-old WT(Col-0) and scap1 mutant plants and extracted RNA independently. Four biological replicates were performed for each experiment.

2013-02-28 | E-GEOD-43964 | biostudies-arrayexpress

Anionic phospholipids connect barley ROP signaling with resistance and susceptibility to fungal cell entry

Project description:Rho-of-plant small GTPases (ROPs) are regulators of cell polar growth processes and of plant-pathogen interactions. The barley ROP protein RACB is involved in susceptibility towards infection by the barley powdery mildew fungus Blumeria hordei, but little is known about the cellular pathways that connect RACB-signaling to disease susceptibility. We now used an untargeted co immunoprecipitation-liquid chromatography-coupled mass spectrometry screening from Bh-infected epidermal layers of barley plants, which expressed green fluorescing protein-tagged constitutively activated (CA) RACB. This uncovered novel RACB interaction partners of plant and fungal origin. Three of those proteins, a plant phosphoinositide phosphatase, a plant phosphoinositide phospholipase and one putative Bh-effector protein appear to be involved in the barley-Bh-pathosystem and function in disease resistance or susceptibility, respectively. RACB and its plant interactors bind to overlapping anionic phospholipid species in vitro, and in case of RACB, this lipid-interaction is mediated by its carboxy-terminal polybasic region (PBR). Four diverse fluorescent plant anionic phospholipid-markers show altered subcellular distribution during Bh-attack. Phosphatidyl-4-phosphate and phosphatidylserine show a distinct enrichment at the haustorial neck region, suggesting a connection to subcellular targeting of RACB at this site. We suggest that the interplay of ROPs with anionic phospholipids and phospholipid–metabolizing enzymes may steer subcellular enrichment of components that are pivotal for fungal penetration success or failure.

2024-12-12 | PXD056811 | Pride

Effect of salinity on the proteome profile of guard cells from the salt-tolerant plant species Beta vulgaris (sugar beet)

Project description:Soil salinity is a major environmental constraint affecting crop growth and threatening global food security. Plants adapt to salinity by optimizing performance of stomata, the microscopic sphincters inserted into the wax-covered epidermis of the shoot, which balance CO2 intake for photosynthetic carbon gain and concomitant water loss. Stomata are formed by two guard cells (GCs) that are morphologically and functionally distinct from the other leaf cells. In order to better understand the molecular mechanisms underlying stomatal function under saline conditions we used proteomics approach to study isolated GCs from the salt-tolerant sugar beet species.

2020-05-27 | PXD017583 | Pride

Proteomic characterisation of the salt gland-enriched tissues of the mangrove tree species Avicennia officinalis

Project description:Plant salt glands are nature’s desalination devices that harbour potentially useful information pertaining to salt and water transport during secretion. As part of the program toward deciphering secretion mechanisms in salt glands, we adopted a shotgun approach to look into the proteome of salt-gland enriched tissues of the mangrove tree species Avicennia officinalis. To achieve this, we isolated the adaxial epidermal peels (which harbour the salt glands), and separated them from the mesophyll tissues of the leaves. Three biological replicates were prepared and total proteins were extracted from these tissues. Proteins that were found to be unique in salt gland-enriched tissues were obtained by eliminating proteins found in the mesophyll tissues. The proteins that are uniquely present in salt gland-enriched tissues were then selected and categorized by GO analysis.

2015-07-22 | PXD000771 | Pride

Transcription profiling of individual resistant and infected epidermalsingle cells of the susceptible barley line Pallas

Project description:Investigation of the interaction between barley and B. graminis. Individual resistant and infected epidermal cells of the susceptible barley line Pallas were easily distinguished by microscopy, and their contents collected in micro-capillaries. We hybridized 12 arrays with radioactively labeled material from these cell specific samples; 3 with material from resistant cells, 3 with material from infected cells, 3 with material from non-inoculated control cells, and 3 identical samples for technical replication. Pooling equal amounts of material from the 3 infected samples made these last 3 samples.

2005-11-13 | E-MEXP-457 | biostudies-arrayexpress

Transcription profiling by array of barley after inoculation with an adapted and a non-adapted Magnaporthe isolate or mock-inoculation to identify candidate genes for nonhost resistance against blast disease

Project description:In this experiment the transcriptome reprogramming in barley during host and nonhost interaction with Magnaporthe sp. was analyzed in a time-series approach. Seven days old barley plants of cv. Vada were mock-inoculated or inoculated with adapted Magnaporthe isolate TH6772 from rice or non-adapted isolate CD180 from Pennisetum. After 6, 12, 24 and 48 hours the abaxial epidermis was sampled. Total RNA was extracted using the RNeasy Plant Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany), and hybridized to Agilent 44k oligonucleotide arrays.

2017-12-02 | E-MTAB-5634 | biostudies-arrayexpress

Expression profiles of bladder and stalk cell enriched epidermal peels from Chenopdium quinoa

Project description:Quinoa, like a large fraction of halophytes, use so-called bladder cells to detoxify excess salt. These leaf exterior structures are formed by specialized trichomes and consist of a leaf epidermal cell, a stalk cell and the bladder cell itself. Under salt stress, Na+ and Cl- as well as K+ and metabolites are imported from leaf sources and stored in the bladder. During this process the stalk cell simultaneously operates as both a selectivity filter and a flux controller. We submerged detached leaves in buffer, then lightly brushed the abaxial surface with a fine paintbrush to remove only the EBCs. The abaxial epidermis was then removed using tweezers and sampled in buffer for RNA extraction and RNA sequencing to separate the gene expression profiles of stalk cells from bladders and leaves.

2022-04-01 | E-MTAB-10363 | biostudies-arrayexpress

Barley Systemic Immunity: Dataset Local and Systemic

Project description:Leaf-to-leaf, systemic immune signaling known as systemic acquired resistance (SAR) is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to SAR in Arabidopsis thaliana, systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid (SA). Instead, we documented a moderate local but not systemic induction of abscisic acid (ABA) after infection of leaves with Psj. In contrast to SA or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or ABA triggered systemic immunity to Xtc. RNA-seq analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest and quantitative RT-PCR associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors possibly facilitating transcriptional reprogramming to potentiate immunity.

2014-11-24 | E-MTAB-3063 | biostudies-arrayexpress

MPK4 interacting proteins in guard cells through AP-MS

Project description:Plants are often challenged by numerous biotic and abiotic environmental stresses. They have evolved efficient signaling networks in response to the environmental challenges, such as pathogen invasion, drought, CO2 changes. Guard cells are specialized epidermal cells enclosing pores on leaf surface, aka, stomata. Stomatal guard cells are at the frontline of biotic and abiotic stress responses. The opening and closing of stomata are regulated by a sophisticated intracellular signaling networks. Mitogen-activated protein kinase 4 (MPK4) was first identified as negative regulator in systemic acquired resistance (SAR). It was also play important roles in cytokinesis, reproduction, and photosynthesis. MPK4 has higher expression levels in plant guard cells than in mesophyll cells. Arabidopsis mpk4 mutant has higher levels of salicylic acid (SA) and reactive oxygen species (ROS), which are related with stomatal movement. The specific function of MPK4 in guard cells is unknown. In order to understand MPK4 functions in guard cells, it is essential to investigate MPK4-associated protein complex. Here we focus on identification of guard cell specific MPK4 complex using affinity purification following by mass spectrometry (AP-MS). Potential substrates were selected and validated with yeast two hybrid (Y2H).

2024-04-30 | PXD034347 | Pride

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