Lipid Flippases and Elemental Homeostasis Systems in Arabidopsis thaliana
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AuthorMcDowell, Stephen C.
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Many molecules in living systems are present in charged forms, and these molecules are often highly regulated. The work presented in the following chapters addresses two main topics involving charged molecules using the model plant <italic>Arabidopsis thaliana</italic>: elemental homeostasis and lipid flippases. The study of elemental homeostasis is referred to as ionomics and is the topic of Chapter II. P<sub>4</sub>-ATPases are thought to be the principle class of proteins with lipid flippase activity and are the topics of Chapter III and Chapter IV. Plants, especially seed crops, are an important source of mineral nutrition in the human diet and are thus important targets for biofortification and toxic element exclusion. Here, we report the results of a pilot ionomic screen in which we quantified the concentrations of 14 elements in Arabidopsis seeds. To identify conditional ionomic phenotypes, plants were grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. To help identify the genetic networks regulating the seed ionome, elemental concentrations were evaluated in mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes observed in the mutant screen supports an estimate that up to 11% of the Arabidopsis genome encodes proteins of functional relevance to the seed ionome. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: <italic>SOS2</italic>, <italic>ABH1</italic>, <italic>CCC</italic>, <italic>At3g14280</italic>, and <italic>CNGC2</italic>. Reproducible ionomic differences were also observed between the Col-0 reference accession and eight of the other nine accessions screened. Significantly, all 15 mutants with reproducible ionomic phenotypes showed at least one change under standard soil conditions. This suggests that the sole use of a standard growth environment might be the most effective strategy for continued reverse-genetic efforts to identify genes that impact the Arabidopsis seed ionome. Nonetheless, each soil modification had a unique impact on the Col-0 seed ionome and elicited several conditional phenotypes in both the mutant and accession screens, indicating that seed elemental homeostasis is sensitive to soil conditions. Together, the results of this study establish that elemental analysis is a sensitive approach to identify genes and environmental conditions that impact elemental accumulation in Arabidopsis seed. By flipping lipids between membrane leaflets, P<sub>4</sub>-ATPases are thought to help create and maintain asymmetry in biological membranes. Lipid asymmetry between membrane leaflets has been implicated in a wide range of biological processes including: vesicular trafficking, cell signaling, modulation of membrane permeability, protein recruitment, and regulation of protein activity. Additionally, one P<sub>4</sub>-ATPase, Neo1p, is essential in yeast. In <italic>Arabidopsis thaliana</italic>, 12 P<sub>4</sub>-ATPases have been identified: <underline>A</underline>minophospho<underline>l</underline>ipid <underline>A</underline>TPase 1 (ALA1) to ALA12. However, very little is known about P<sub>4</sub>-ATPases in the context of plant systems. Of the 12 ALA isoforms, only ALA3 has been extensively studied. Previous studies have shown that loss of ALA3 results in pleiotropic phenotypes affecting root, shoot, and reproductive development. Here, we expand on the previous studies by showing that multiple phenotypes for <italic>ala3</italic> mutants are strongly sensitive to growth conditions. We also expand on the <italic>ala3</italic> pollen phenotype by identifying three points of defect in <italic>ala3</italic> pollen tubes: delayed germination, slow growth, and reduced overall length. Furthermore, we show that <italic>ala3</italic> pistils have reduced ovule production, thus providing the first evidence of a female reproductive defect in <italic>ala3</italic> mutants. Together, these results support a model in which ALA3 functions in multiple cell types and is critical to plants for development and adaptation to varied growth conditions. Two other ALA isoforms, ALA6 and ALA7, were also examined in this study. We provide <italic>in-vitro</italic> and <italic>in-vivo</italic> evidence that ALA6 and ALA7 are important for rapid, sustained pollen tube growth. Expression of fluorescently labeled ALA6 fusion proteins indicates that the subcellular localization of ALA6 includes the plasma membrane and highly mobile endomembrane structures. We also show that staining by lipophilic FM dyes is reduced by ~10-fold in <italic>ala6-1/7-2</italic> pollen tubes relative to wild-type, suggesting differences in plasma membrane composition. Furthermore, tandem mass spectroscopy analysis revealed significant differences between the lipid compositions of <italic>ala6-1/7-2</italic> and wild-type pollen grains, both in the concentrations of different headgroups and in the average number of double bonds present within acyl side chains. Together, these results support a model in which ALA6 and ALA7 function to directly or indirectly regulate the distribution and concentration of lipids in pollen and are thus critical for pollen fitness.