Transcriptomic Profiling of the Arabidopsis Embryonic Epidermis Using FANS in Combination with RNAseq.

The fundamental mechanisms of cell identity and tissue establishment are important already from the very beginning of a plant's life and reiterate later during development. In order to unravel and understand the underlying mechanisms to generate differences that in turn lead to cell or tissue types, plant cells have to be separated and their transcriptional setup analyzed. We have previously demonstrated that fluorescence-activated nuclear sorting (FANS) is a powerful tool to generate nuclear transcriptomic profiles of the most inaccessible embryonic tissues. In this protocol, we extend this effort to combine FANS with next generation RNA sequencing (RNA-seq) to achieve early embryonic transcriptomes of Arabidopsis epidermis precursor tissue (protoderm) and the inner tissue counterpart.

Identification of two-component system elements downstream of AHK5 in the stomatal closure response of Arabidopsis thaliana.

To optimize water use efficiency, plants regulate stomatal closure through a complex signaling process. Hydrogen peroxide (H2O2) is produced in response to several environmental stimuli, and has been identified as a key second messenger involved in the regulation of stomatal aperture. The Arabidopsis histidine kinase 5 (AHK5) has been shown to regulate stomatal closure in response to H2O2 and other stimuli that depend on H2O2. AHK5 is a member of the two-component system (TCS) in Arabidopsis. The plant TCS comprises three different protein types: the hybrid histidine kinases (HKs), the phosphotransfer proteins (HPs) and the response regulators (RRs). Here we determined TCS elements involved in H2O2- and ethylene-dependent stomatal closure downstream of AHK5. By yeast and in planta interaction assays and functional studies, AHP1, 2 and 5 as well as the response regulators ARR4 and ARR7 were identified acting downstream of AHK5 in the ethylene and H2O2 response pathways of guard cells. Furthermore, we demonstrate that aspartate phosphorylation of ARR4 is only required for the H2O2- but not for the ethylene-induced stomatal closure response. Our data suggest the presence of a complex TCS signaling network comprising of at least AHK5, several AHPs and response regulators, which modulate stomatal closure in response to H2O2 and ethylene.

Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum.

Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants.

The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation.

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarisation of the plasma membrane which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarisation and wall expansion, the activation of the plasma membrane P-type H+-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyse long-distance BL signaling in Arabidopsis seedlings.

A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana.

To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion.

Latest news on Arabidopsis brassinosteroid perception and signaling.

Brassinosteroids (BRs) are plant hormones regulating growth and development. In interaction with other hormones, they are involved in environmental cue responses. The present model of the BR response pathway in Arabidopsis includes the perception of the hormone by the plasma membrane (PM) receptor brassinosteroid insensitive 1 (BRI1) and its hetero-oligomerization with the co-receptor BRI1-associated receptor kinase 1 (BAK1), followed by the activation of a signaling-cascade finally resulting in the expression of BR-responsive genes. New findings have shed light on the receptor density in the PM and on the molecular mechanism of BR perception, which includes the hormone-induced formation of a platform in the BRI1 extracellular domain for interaction with BAK1. Furthermore, new knowledge on early, BRI1-initiated signaling events at the PM-cytoplasm interface has recently been gained. In addition, a fast BR response pathway that modifies the membrane potential and the expansion of the cell wall - both crucial processes preceding cell elongation growth - have been identified. In this review, these latest findings are summarized and discussed against the background of the present model of BRI1 signaling.

The histidine kinase AHK5 integrates endogenous and environmental signals in Arabidopsis guard cells.

BACKGROUND: Stomatal guard cells monitor and respond to environmental and endogenous signals such that the stomatal aperture is continually optimised for water use efficiency. A key signalling molecule produced in guard cells in response to plant hormones, light, carbon dioxide and pathogen-derived signals is hydrogen peroxide (H(2)O(2)). The mechanisms by which H(2)O(2) integrates multiple signals via specific signalling pathways leading to stomatal closure is not known. PRINCIPAL FINDINGS: Here, we identify a pathway by which H(2)O(2), derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure. Histidine kinases (HK) are part of two-component signal transduction systems that act to integrate environmental stimuli into a cellular response via a phosphotransfer relay mechanism. There is little known about the function of the HK AHK5 in Arabidopsis thaliana. Here we report that in addition to the predicted cytoplasmic localisation of this protein, AHK5 also appears to co-localise to the plasma membrane. Although AHK5 is expressed at low levels in guard cells, we identify a unique role for AHK5 in stomatal signalling. Arabidopsis mutants lacking AHK5 show reduced stomatal closure in response to H(2)O(2), which is reversed by complementation with the wild type gene. Over-expression of AHK5 results in constitutively less stomatal closure. Abiotic stimuli that generate endogenous H(2)O(2), such as darkness, nitric oxide and the phytohormone ethylene, also show reduced stomatal closure in the ahk5 mutants. However, ABA caused closure, dark adaptation induced H(2)O(2) production and H(2)O(2) induced NO synthesis in mutants. Treatment with the bacterial pathogen associated molecular pattern (PAMP) flagellin, but not elf peptide, also exhibited reduced stomatal closure and H(2)O(2) generation in ahk5 mutants. SIGNIFICANCE: Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H(2)O(2) homeostasis and is independent of ABA signalling in guard cells.