Site-directed genotype screening for elimination of antinutritional saponins in quinoa seeds identifies TSARL1 as a master controller of saponin biosynthesis selectively in seeds.

Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait. We mapped genes controlling seed saponin content to a genomic region that includes TSARL1. We isolated desired genetic variation in this gene by producing a large mutant library of a commercial quinoa cultivar and screening the library for specific nucleotide substitutions using droplet digital PCR. We were able to rapidly isolate two independent tsarl1 mutants, which retained saponins in the leaves and roots for defence, but saponins were undetectable in the seed coat. We further could show that TSARL1 specifically controls seed saponin biosynthesis in the committed step after 2,3-oxidosqualene. Our work provides new important knowledge on the function of TSARL1 and represents a breakthrough for quinoa breeding.

Lipid flippases in polarized growth.

Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.

The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase.

Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2-ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.

Plant P4-ATPase lipid flippases: How are they regulated?

P4 ATPases are active membrane transporters that translocate lipids towards the cytosolic side of the biological membranes in eukaryotic cells. Due to their essential cellular functions, P4 ATPase activity is expected to be tightly controlled, but fundamental aspects of the regulation of plant P4 ATPases remain unstudied. In this mini-review, our knowledge of the regulatory mechanisms of yeast and mammalian P4 ATPases will be summarized, and sequence comparison and structural modelling will be used as a basis to discuss the putative regulation of the corresponding plant lipid transporters.

Mini-review: Lipid flippases as putative targets for biotechnological crop improvement.

An increasing world population and drastic changes in weather conditions are challenging agricultural production. To face these challenges and ensure sustainable food production in the future, crop plants need to be improved to withstand several different biotic and abiotic stresses. Commonly, breeders select varieties that can tolerate a specific type of stress and then cross these varieties to stack beneficial traits. This strategy is time-consuming and strictly dependent on the stacked traits been genetically unlinked. Here, we revise the role of plant lipid flippases of the P4 ATPase family in stress-related responses with a special focus on the pleiotropic nature of their functions and discuss their suitability as biotechnological targets for crop improvement.

Plant transbilayer lipid asymmetry and the role of lipid flippases.

Many biological membranes present an asymmetric lipid distribution between the two leaflets that is known as the transbilayer lipid asymmetry. This asymmetry is essential for cell survival and its loss is related to apoptosis. In mammalian and yeast cells, ATP-dependent transport of lipids to the cytosolic side of the biological membranes, carried out by so-called lipid flippases, contributes to the transbilayer lipid asymmetry. Most of these lipid flippases belong to the P4-ATPase protein family, which is also present in plants. In this review, we summarize the relatively scarce literature concerning the presence of transbilayer lipid asymmetry in different plant cell membranes and revise the potential role of lipid flippases of the P4-ATPase family in generation and/or maintenance of this asymmetry.

Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p.

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.

Catch You on the Flip Side: A Critical Review of Flippase Mutant Phenotypes.

Lipid flippases are integral membrane proteins that use ATP hydrolysis to power the generation of phospholipid asymmetry between the two leaflets of biological membranes, a process essential for cell survival. Although the first report of a plant lipid flippase was published in 2000, progress in the field has been slow, partially due to the high level of redundancy in this gene family. However, recently an increasing number of reports have examined the physiological function of lipid flippases, mainly in Arabidopsis thaliana. In this review we aim to summarize recent findings on the physiological relevance of lipid flippases in plant adaptation to a changing environment and caution against misinterpretation of pleiotropic effects in genetic studies of flippases.

The P5A ATPase Spf1p is stimulated by phosphatidylinositol 4-phosphate and influences cellular sterol homeostasis.

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER.

The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation.

Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P(4)-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.