Modulation of ABA responses by the protein kinase WNK8.

Abscisic acid (ABA) regulates growth and developmental processes in response to limiting water conditions. ABA functions through a core signaling pathway consisting of PYR1/PYL/RCAR ABA receptors, type 2C protein phosphatases (PP2Cs), and SnRK2-type protein kinases. Other signaling modules might converge with ABA signals through the modulation of core ABA signaling components. We have investigated the role of the protein kinase WNK8 in ABA signaling. WNK8 interacted with PP2CA and PYR1, phosphorylated PYR1 in vitro, and was dephosphorylated by PP2CA. A hypermorphic wnk8-ct Arabidopsis mutant allele suppressed ABA and glucose hypersensitivities of pp2ca-1 mutants during young seedling development, and WNK8 expression in protoplasts suppressed ABA-induced reporter gene expression. We conclude that WNK8 functions as a negative modulator of ABA signaling.

Copper status of exposed microorganisms influences susceptibility to metallic nanoparticles.

Although interactions of metallic nanoparticles (NPs) with various microorganisms have been previously explored, few studies have examined how metal sensitivity impacts NP toxicity. The present study investigated the effects of copper NPs (Cu-NP) exposure on the model alga Chlamydomonas reinhardtii in the presence and absence of the essential micronutrient copper. The toxic ranges for Cu-NPs and the ionic control, CuCl2 , were determined using a high-throughput adenosine triphosphate (ATP)-based fluorescence assay. The Cu-NPs caused similar mortality in copper-replete and copper-deplete cells (median inhibitory concentration [IC50]: 14-16 mg/L) but were less toxic than the ionic control, CuCl2 (IC50: 7 mg/L). Using this concentration range, the Cu-NP impacts on cell morphology, copper accumulation, chlorophyll content, and expression of stress genes under both copper supply states were assessed. Osmotic swelling, membrane damage, and chloroplast and organelle disintegration were observed by transmission electron microscopy at both conditions. Despite these similarities, copper-deplete cells showed greater accumulation of loosely bound and tightly bound copper after exposure to Cu-NPs. Furthermore, copper-replete cells experienced greater loss of chlorophyll content, 19% for Cu-NPs, compared with only an 11% net decrease in copper-deplete cells. The tightly bound copper was bioavailable as assessed by reverse-transcriptase quantitative polymerase chain reaction analysis of CYC6, a biomarker for Cu deficiency. The increased resistance of copper-deplete cells to Cu-NPs suggests that these cells potentially metabolize excess Cu-NPs or better manage sudden influxes of ions. The results suggest that toxicity assessments must account for the nutritional status of impacted organisms and use toxicity models based on estimations of the bioavailable fractions.

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

Remodeling of membrane lipids in iron-starved Chlamydomonas.

Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.

A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii.

Interest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutner's supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni²âº-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni²âº because of the introduction of an EDTA buffer system in the revised medium. Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology.

Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis.

In eukaryotic cells, compartments of the highly dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H(+)-ATPases (V-ATPases). In the Arabidopsis thaliana genome, most V-ATPase subunits are encoded by small gene families, thus offering potential for a multitude of enzyme complexes with different kinetic properties and localizations. We have determined the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a. Colocalization experiments as well as immunogold labeling showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocks the endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Furthermore, nascent cell plates are rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Together, our results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.

A WNK kinase binds and phosphorylates V-ATPase subunit C.

WNK (with no lysine (K)) protein kinases are found in many eukaryotes and share a unique active site. Here, we report that a member of the Arabidopsis WNK family (AtWNK8) interacts with subunit C of the vacuolar H+-ATPase (V-ATPase) via a short C-terminal domain. AtWNK8 is shown to autophosphorylate intermolecularly and to phosphorylate Arabidopsis subunit C (AtVHA-C) at multiple sites as determined by MALDI-TOF MS analysis. Furthermore, we show that AtVHA-C and other V-ATPase subunits are phosphorylated when V1-complexes are used as substrates for AtWNK8. Taken together, our results provide evidence that V-ATPases are potential targets of WNK kinases and their associated signaling pathways.