Genetically encoded fluorescent sensors adapted to acidic pH highlight subdomains within the plant cell apoplast.

Monitoring pH is one of the challenges in understanding diverse physiological regulations as well as ionic balance, especially in highly acidic environments such as the apoplast and the vacuole. To circumvent the poor efficiency of pH measurements below pH 5, we designed three genetically encoded sensors composed of two fluorescent proteins in tandem. We selected fluorescent protein pairs of low but sufficiently different pKa so that each protein could differentially sense the imposed pH.

pH biosensing in the plant apoplast-a focus on root cell elongation.

The pH parameter of soil plays a key role for plant nutrition as it is affecting the availability of minerals and consequently determines plant growth. Although the mechanisms by which root perceive the external pH is still unknown, the impact of external pH on tissue growth has been widely studied especially in hypocotyl and root. Thanks to technological development of cell imaging and fluorescent sensors, we can now monitor pH in real time with at subcellular definition.

Adjustment of K Fluxes and Grapevine Defense in the Face of Climate Change.

Grapevine is one of the most economically important fruit crops due to the high value of its fruit and its importance in winemaking. The current decrease in grape berry quality and production can be seen as the consequence of various abiotic constraints imposed by climate changes. Specifically, produced wines have become too sweet, with a stronger impression of alcohol and fewer aromatic qualities.

Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging.

Building a proton gradient across a biological membrane and between different tissues is a matter of great importance for plant development and nutrition. To gain a better understanding of proton distribution in the plant root apoplast as well as across the plasma membrane, we generated plants expressing stable membrane-anchored ratiometric fluorescent sensors based on pHluorin. These sensors enabled noninvasive pH-specific measurements in mature root cells from the medium-epidermis interface up to the inner cell layers that lie beyond the Casparian strip.

Vacuolar Trafficking Protein VPS38 Is Dispensable for Autophagy.

Phosphatidylinositol 3-P (PI3P) is a signaling molecule that controls a variety of processes in endosomal, autophagic, and vacuolar/lysosomal trafficking in yeasts and mammals. Vacuolar protein sorting 34 (Vps34) is a conserved PI3K present in multiple complexes with specific functions and regulation. In yeast, the PI3K complex II consists of Vps34p, Vps15p, Vps30p/Atg6p, and Vps38p, and is essential for vacuolar protein sorting.

pH Regulation by NHX-Type Antiporters Is Required for Receptor-Mediated Protein Trafficking to the Vacuole in Arabidopsis.

Protein trafficking requires proper ion and pH homeostasis of the endomembrane system. The NHX-type Na(+)/H(+) antiporters NHX5 and NHX6 localize to the Golgi, trans-Golgi network, and prevacuolar compartments and are required for growth and trafficking to the vacuole. In the nhx5 nhx6 T-DNA insertional knockouts, the precursors of the 2S albumin and 12S globulin storage proteins accumulated and were missorted to the apoplast.

Development and properties of genetically encoded pH sensors in plants.

Fluorescent proteins (FPs) have given access to a large choice of live imaging techniques and have thereby profoundly modified our view of plant cells. Together with technological improvement of imaging, they have opened the possibility to monitor physico-chemical changes within cells. For this purpose, a new generation of FPs has been engineered.

In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.

Plant defensin AhPDF1.1 is not secreted in leaves but it accumulates in intracellular compartments.

• Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. • Stable and transient expression of AhPDF1.

The cytosolic tail dipeptide Ile-Met of the pea receptor BP80 is required for recycling from the prevacuole and for endocytosis.

Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane.

The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development.

Background: PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na(+)-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Expression of a glycosylated GFP as a bivalent reporter in exocytosis.

The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an alpha-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a beta-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells.

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide.

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides.

AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells.

Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach.

Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters.

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins.

Arabidopsis mu A-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1.

In receptor-mediated transport pathways in mammalian cells, clathrin-coated vesicle (CCV) mu-adaptins are the main binding partners for the tyrosine sorting/internalization motif (YXXØ). We have analyzed the function of the mu A-adaptin, one of the five mu-adaptins from Arabidopsis thaliana, by pull-down assays and plasmon resonance measurements using its receptor-binding domain (RBD) fused to a histidine tag. We show that this adaptin is able to bind the consensus tyrosine motif YXXØ from the pea vacuolar sorting receptor (VSR)-PS1, as well as from the mammalian trans-Golgi network (TGN)38 protein.

The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain.

The tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain.