Uses and misuses of the fudge factor in quantitative discovery proteomics

Selecting proteins with significant differential abundance is the cornerstone of many relative quantitative proteomics experiments. To do so, a trade‐off between p‐value thresholding and fold‐change thresholding can be performed because of a specific parameter, named fudge factor, and classically noted s₀. We have observed that this fudge factor is routinely turned away from its original (and statistically valid) use, leading to important distortion in the distribution of p‐values, jeopardizing the protein differential analysis, as well as the subsequent biological conclusion. In this article, we provide a comprehensive viewpoint on this issue, as well as some guidelines to circumvent it.     

Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.     

An ectomycorrhizal fungus alters sensitivity to jasmonate,salicylate, gibberellin,and ethylene in host roots

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography–tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone-treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid-stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development.     

4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.     

Cross-talk between aldosterone and angiotensin signaling in vascular smooth muscle cells

In hypertension or other forms of cardiovascular disease, the chronic activation of the renin-angiotensin-aldosterone system (RAAS) leads to dysfunction of the vasculature, including, increased vascular tone, inflammation, fibrosis and thrombosis. Cross-talk between the main mediators of the RAAS, aldosterone and angiotensin (Ang) II, participates in the development of this vascular dysfunction. Recent studies have highlighted the molecular mechanisms supporting this cross-talk in vascular smooth muscle cells (VSMCs). Some of the signaling pathways activated by the Ang II type 1 receptor (AT₁R) are dependent on the mineralocorticoid receptor (MR) and vice versa. VSMC signaling pathways involved in migration and growth are under the control of cross-talk between aldosterone and Ang II. A synergistic mechanism leads to potentiation of signaling pathways activated by each agent. The genomic and non-genomic mechanisms activated by aldosterone cooperate with Ang II to regulate vascular tone and gene expression of pro-inflammatory and pro-fibrotic molecules. This cross-talk is dependent on the non-receptor tyrosine kinase c-Src, and on receptor tyrosine kinases, EGFR and PDGFR, and leads to activation of MAP kinases and growth, migration and inflammatory effects. These new findings will contribute to development of better treatments for conditions in which the RAAS is excessively activated.     

Fluorescent in situ visualization of sterols in Arabidopsis roots

Sterols are eukaryotic membrane components with crucial roles in diverse cellular processes. Elucidation of sterol function relies on development of tools for in situ sterol visualization. Here we describe protocols for in situ sterol localization in Arabidopsis thaliana root cells, using filipin as a specific probe for detection of fluorescent filipin-sterol complexes. Currently, filipin is the only established tool for sterol visualization in plants. Filipin labeling can be performed on aldehyde-fixed samples, largely preserving fluorescent proteins and being compatible with immunocytochemistry. Filipin can also be applied for probing live cells, taking into account the fact that it inhibits sterol-dependent endocytosis. The experimental procedures described are designed for fluorescence detection by confocal laser-scanning microscopy with excitation of filipin-sterol complexes at 364 nm. The protocols require 1 d for sterol covisualization with fluorescent proteins in fixed or live roots and 2 d for immunocytochemistry on whole-mount roots.     

Mechanisms of auxin-dependent cell and tissue polarity

The establishment of cellular asymmetries and their coordination within the tissue layer are fundamental to the development of multicellular organisms. In plants, the induction and coordination of cell polarity have classically been attributed to involve the hormone auxin and its flow. However, the underlying mechanisms have only recently been addressed at the molecular level. We review progress on the characterisation of the auxin influx and efflux carrier properties of specific plasma membrane proteins, mechanisms underlying their delivery to and internalisation from the plasma membrane, their endocytic transport and degradation. We discuss mechanisms of auxin gradient, transport and response action during the coordination of polarity, along with the downstream involvement of Rho-of-plant small GTPases during the execution of cell polarity.