Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

Manipulation of Mitogen-Activated Protein Kinase Kinase Signaling in the Arabidopsis Stomatal Lineage Reveals Motifs That Contribute to Protein Localization and Signaling Specificity

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four “stomatal” MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.     

Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium,Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling

The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)–dependent protein kinases, but symbiotic signaling involves a calcium/CaM–dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.     

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.     

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.Membrane trafficking is essential in eukaryotic cells. Cellular membranes serve as a delivery system for newly synthesized proteins such as transporters and receptors exiting the endoplasmic reticulum after proper folding. They then transit through the Golgi complex, reaching the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. Receptors and transporters returning from the PM are either recycled or targeted to the vacuole for degradation. Delivery and recycling sorting pathways overlap in the trans-Golgi network (TGN)/early endosome (EE), an intermediate compartment for both exocytosis and endocytosis (Reyes et al., 2011). In plant systems, the endoplasmic reticulum and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD), cytoplasmic channels that regulate traffic in the symplasm (Maule et al., 2011).The selective transport of macromolecules between different compartments of the endomembrane system is mediated by coat proteins promoting the generation of small cargo-trafficking coated vesicles (Spang, 2008). The recognition and recruitment of cargo proteins are mediated by so-called adaptor complexes (AP complexes [AP-1–AP-4]; Robinson, 2004) one of which, AP-1, is localized on the TGN/EE and endosomes, whereas AP-2 is in the PM. The μ-subunit of AP complexes is devoted to cargo protein selection via a specific and well-characterized interaction with a Tyr-sorting signal, YXXΦ, where Φ is a bulky hydrophobic residue and X is any amino acid (Bonifacino and Dell’Angelica, 1999). YXXΦ motifs are present in the cytoplasmic tail of many proteins integral to the PM and TGN/EE and have been found in the movement proteins (MPs) of some viruses (Laporte et al., 2003; Haupt et al., 2005). Plant viruses are obligate parasites that exploit host components to move within the cell and from cell to cell into the vascular system for systemic invasion of the host. Virus movement, which requires the passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes (Harries et al., 2010). While most MPs act to increase the size exclusion limit of PD to facilitate the passage of the viral nucleoprotein complex, other MPs are assembled in tubules that pass inside highly modified PD and transport encapsidated particles through their lumen.Here, we focus on this second group of tubule-forming MPs and examine the intracellular trafficking of cauliflower mosaic virus (CaMV) MP. The MP encoded by CaMV forms tubules guiding encapsidated virus particle cell-to-cell transport via an indirect MP-virion interaction (Stavolone et al., 2005; Sánchez-Navarro et al., 2010). However, how CaMV MP (and the other tubule-forming MPs) targets the PM and forms tubules remains to be elucidated. Tubule-forming MPs do not require an intact cytoskeleton for PM targeting (Huang et al., 2000; Pouwels et al., 2002) and/or tubule formation (Laporte et al., 2003). However, suppression of tubule formation upon treatment with brefeldin A (BFA), a specific inhibitor of secretion or endocytosis, suggests the involvement of the endomembrane system in correct functioning of some tubule-forming MPs (Huang et al., 2000; Laporte et al., 2003). In this study, we examined the three Tyr-sorting motifs in CaMV MP and show that each of the three domains interacts directly with subunit μ of an Arabidopsis (Arabidopsis thaliana) AP complex. Mutations in these domains revert in the viral context to maintain CaMV viability. MP is found in endosomal compartments labeled by AtRAB-F2b (ARA7) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). The presence of at least one functional YXXΦ domain is essential for the localization of MP to endosomes and for tubule assembly but is not required for MP targeting to the PM. We provide several lines of evidence to show CaMV MP trafficking in the endocytic pathway. Our findings are discussed in the light of the recent demonstration that the TGN/EE functions as a major hub controlling secretory and endocytic pathways in plants.     

Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and ERO1 in Soybean

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a′ domain among the a, a′, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a′ domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a′ domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants.In eukaryotes, many secretory and membrane proteins fold via disulfide bond formation in the endoplasmic reticulum (ER). Seed storage proteins of major crops, such as wheat, corn, rice, and beans, which are important protein sources for humans and domestic animals, are synthesized in the ER of the endosperm or cotyledon. A number of seed storage proteins fold by the formation of intramolecular disulfide bonds (oxidative folding) and are transported to and accumulate in protein bodies (Kermode and Bewley, 1999; Jolliffe et al., 2005). In contrast to normally folded proteins, misfolded and unfolded proteins are retained in the ER and degraded by an ER-associated degradation or vacuolar system (Smith et al., 2011; Pu and Bassham, 2013). Therefore, quick and efficient oxidative folding of nascent seed storage proteins is needed for their accumulation in protein bodies.During this process, protein disulfide isomerase (PDI; EC 5.3.4.1) and other ER protein thiol disulfide oxidoreductases (ER oxidoreductases) are thought to catalyze the formation and isomerization of disulfide bonds in nascent proteins (Hatahet and Ruddock, 2009; Feige and Hendershot, 2011; Lu and Holmgren, 2014). After phylogenetic analysis of the Arabidopsis genome, 10 classes of ER oxidoreductases (classes I–X) were identified (Houston et al., 2005). Among them, class I ER oxidoreductase, a plant PDI ortholog, has been studied in a wide variety of plants. Class I ER oxidoreductases have two catalytically active domains a and a′, containing active centers composed of Cys-Gly-His-Cys and two catalytically inactive domains b and b′. An Arabidopsis ortholog of class I ER oxidoreductases is required for proper seed development and regulates the timing of programmed cell death by chaperoning and inhibiting Cys proteases (Andème Ondzighi et al., 2008). OaPDI, a PDI from Oldenlandia affinis, a coffee family (Rubiaceae) plant, is involved in the folding of knotted circular proteins (Gruber et al., 2007). The rice ortholog (PDIL1-1) was suggested to be involved in the maturation of the major seed storage protein glutelin (Takemoto et al., 2002). Furthermore, rice PDIL1-1 plays a role in regulatory activities for various proteins that are essential for the synthesis of grain components as determined by analysis of a T-DNA insertion mutant (Satoh-Cruz et al., 2010).The oxidative refolding ability of class I ER oxidoreductases was confirmed in recombinant soybean (GmPDIL-1) and wheat proteins produced by an Escherichia coli expression system established from cDNAs (Kamauchi et al., 2008; Kimura et al., 2015).Class II and III ER oxidoreductases have an a–b–b′–a′ domain structure. Class II ER oxidoreductases have an acidic amino acid-rich sequence in the N-terminal region ahead of the a domain. Recombinant soybean (GmPDIL-2) and wheat class II ER oxidoreductases have oxidative refolding activities similar to that of class I (Kamauchi et al., 2008; Kimura et al., 2015). Class III ER oxidoreductases contain the nonclassical redox-center Cys-X-X-Ser/Cys motifs, as opposed to the more traditional CGHC sequence, in the a and a′ domains. Recombinant soybean (GmPDIL-3) and wheat proteins lack oxidative refolding activity in vitro (Iwasaki et al., 2009; Kimura et al., 2015). Class IV ER oxidoreductases are unique to plants and have an a–a′–ERp29 domain structure, which is homologous to the C-terminal domain of mammalian ERp29 (Demmer et al., 1997).Recombinant soybean class IV ER oxidoreductases (GmPDIS-1 and GmPDIS-2) and wheat class IV ER oxidoreductase possess an oxidative refolding activity that is weaker than that of classes I and II (Wadahama et al., 2007; Kimura et al., 2015). Class V ER oxidoreductases are plant orthologs of mammalian P5 and have an a–a′–b domain structure. A rice class V ER oxidoreductase, consisting of PDIL2 and PDIL3, plays an important role in the accumulation of the seed storage protein Cys-rich 10-kD prolamin (crP10; Onda et al., 2011). Recombinant soybean class V ER oxidoreductase, GmPDIM and wheat class V ER oxidoreductase possess an oxidative refolding activity similar to that of class IV (Wadahama et al., 2008; Kimura et al., 2015). In the soybean, GmPDIL-1, GmPDIL-2, GmPDIM, GmPDIS-1, and GmPDIS-2 were found to associate transiently with a seed storage precursor protein, proglycinin, in the ER of the cotyledon by coimmunoprecipitation experiments, suggesting that multiple ER oxidoreductases are involved in the folding of the nascent proglycinin.The disulfide bond in the active center of ER oxidoreductases is reduced as a result of catalyzing disulfide bond formation in an unfolded protein. The reduced active center of PDI was discovered to be oxidized again by ER oxidoreductin-1 (Ero1p) in yeast (Frand and Kaiser, 1998; Pollard et al., 1998). Ero1p orthologs are present universally in eukaryotes. Yeast and flies have a single copy of the ERO1 gene, which is essential for survival (Frand and Kaiser, 1998; Pollard et al., 1998; Tien et al., 2008). Mammals have two genes encoding Ero1-α (Cabibbo et al., 2000) and Ero1-β (Pagani et al., 2000) that function as major disulfide donors to nascent proteins in the ER, but are not critical for survival (Zito et al., 2010). Domain a of yeast PDI is the most favored substrate of yeast Ero1p (Vitu et al., 2010), whereas a′ of human PDI is specifically oxidized by human Ero1-α (Chambers et al., 2010) and Ero1-β (Wang et al., 2011). Electrons from Cys residues of the active centers of PDI are transferred to oxygen by Ero1 (Tu and Weissman, 2004; Sevier and Kaiser, 2008). The reaction mechanisms of yeast Ero1p and human Ero1s have been intensively investigated; their regulation by PDI has been extensively studied as well (Tavender and Bulleid, 2010; Araki and Inaba, 2012; Benham et al., 2013; Ramming et al., 2015). Only rice Ero1 (OsERO1) has been identified as a plant ortholog of Ero1p (Onda et al., 2009). OsERO1 is necessary for disulfide bond formation in rice endosperm. The formation of native disulfide bonds in the major seed storage protein proglutelin was demonstrated to depend upon OsERO1 by RNAi knockdown experiments. However, no plant protein thiol disulfide oxidoreductases that are oxidized by a plant Ero1 ortholog have been identified to date.In this study, we show that multiple soybean ER oxidoreductases can be activated by a soybean Ero1 ortholog (GmERO1a). In addition, we propose a synergistic mechanism by which GmPDIM and GmPDIL-2 cooperatively fold unfolded proteins using oxidizing equivalents provided by GmERO1 in vitro.     

Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated β-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd- and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.Plant cells are interconnected with cross-wall membranous channels called plasmodesmata (Pd). Recent studies have shown that the region of the plasma membrane (PM) lining the Pd channel is a specialized membrane microdomain whose lipid and protein composition differs from the rest of the PM (Tilsner et al., 2011, 2016; Bayer et al., 2014; González-Solís et al., 2014; Grison et al., 2015). In a similar manner, the cell wall domain surrounding the Pd channel is specialized and, unlike the rest of the cell wall, is devoid of cellulose, rich in pectin, and contains callose (an insoluble β-1,3-glucan; Zavaliev et al., 2011; Knox and Benitez-Alfonso, 2014). In response to physiological signals, callose can be transiently deposited and degraded at Pd, which provides a mechanism for controlling the Pd aperture in diverse developmental and stress-related processes (Zavaliev et al., 2011). Control of Pd functioning is mediated by proteins that are specifically targeted to Pd. Plasmodesmal proteins localized to the PM domain of Pd can be integral transmembrane proteins, such as Pd-localized proteins (Thomas et al., 2008), the receptor kinase ARABIDOPSIS CRINKLY4 (Stahl et al., 2013), and callose synthases (Vatén et al., 2011). Alternatively, Pd proteins can associate with the membrane through a lipid modification like myristoylation (e.g. remorins; Raffaele et al., 2009) or be attached by a glycosylphosphatidylinositol (GPI) anchor (e.g. Pd-associated β-1,3-glucanases [BG_pap]; Levy et al., 2007; Rinne et al., 2011; Benitez-Alfonso et al., 2013), Pd-associated callose-binding proteins (PDCBs; Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING PROTEIN2 (LYM2; Faulkner et al., 2013).Among the known Pd proteins involved in Pd-specific callose degradation is BG_pap, a cell wall enzyme carrying a glycosyl hydrolase family 17 (GH17) module as its functional domain (Levy et al., 2007). Another group of proteins controlling callose dynamics at Pd are PDCBs that harbor a callose-binding domain termed carbohydrate-binding module 43 (CBM43), implicated in stabilizing callose at Pd (Simpson et al., 2009). Some β-1,3-glucanases may combine the two callose-modifying activities by harboring both GH17 and CBM43 functional domains, and several such proteins were shown to localize to Pd (Rinne et al., 2011; Benitez-Alfonso et al., 2013; Gaudioso-Pedraza and Benitez-Alfonso, 2014). A distinct feature of BG_pap and PDCBs is that both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs). The GPI anchor is a form of posttranslational modification common to many cell surface proteins in all eukaryotes. GPI-APs are covalently attached to the outer leaflet of the PM through the GPI anchor. The basic structure of the anchor consists of ethanolamine phosphate, followed by a glycan chain of three Man residues and glucosamine, followed by phosphatidylinositol lipid moiety (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6myoinositol-1-P-lipid; Ferguson et al., 2009). All predicted GPI-APs carry an N-terminal secretion signal peptide (SP) similar to other secreted proteins. Distinctly, GPI-APs also carry a structurally conserved 25- to 30-residue C-terminal GPI attachment signal, which typically begins with a small amino acid (e.g. Ala, Asn, Asp, Cys, Gly, or Ser) termed omega, followed by a spacer region of five to 10 polar residues, and ending with a transmembrane segment of 15 to 20 hydrophobic residues (Ferguson et al., 2009). The entire region between the N-terminal and the C-terminal signals of a GPI-AP is termed the ectodomain and carries the protein’s functional domain(s). The GPI modification process takes place in the lumenal face of the endoplasmic reticulum (ER) in a cotranslational manner. Upon translocation into the ER, a GPI-AP is stabilized in the ER membrane by its C-terminal signal, which is concurrently cleaved after the omega amino acid, and a preassembled GPI anchor is covalently attached to the C terminus of the omega amino acid. After attachment to a protein, the GPI anchor undergoes a series of modifications (remodeling), both at the glucan chain and at the lipid moiety. Such remodeling is crucial for the sorting of GPI-APs in the secretory pathway and the subsequent lateral heterogeneity at the PM (Kinoshita, 2015). In particular, the addition of saturated fatty acid chains to the lipid moiety of the anchor leads to the enriched accumulation of GPI-APs in the PM microdomains, also termed lipid rafts (Muñiz and Zurzolo, 2014). In Arabidopsis (Arabidopsis thaliana), GPI modification has been predicted for 210 proteins of diverse functions at the PM or the cell wall or both (Borner et al., 2002). Despite extensive research on the GPI modification pathway and the function of GPI-APs in mammalian and yeast cells, such knowledge in plant systems is scarce. In particular, despite an emerging role of GPI-APs in the regulation of the cell wall domain of Pd, their subcellular targeting and compartmentalization mechanism have not been studied. In addition, it is not known how the targeting mechanism of Pd-resident GPI-APs is different from that of other classes of GPI-APs, which are not localized to Pd.In this study, we investigated the subcellular targeting mechanism of Pd-associated callose-modifying GPI-APs, BG_pap and PDCB1, and compared it with that of two unrelated non-Pd GPI-APs, ARABINOGALACTAN PROTEIN4 (AGP4) and LIPID TRANSFER PROTEIN1 (LTPG1). Using sequential fluorescent labeling of protein domains, we found that the C-terminal GPI modification signal present in both Pd- and non-Pd GPI-APs can function as a primary signal in targeting proteins to the Pd-enriched PM domain. Moreover, we show that while the GPI signal is sufficient for Pd targeting, the ectodomains in BG_pap and PDCB1 further enhance their accumulation at Pd. In contrast, the ectodomains in non-Pd GPI-APs mediate exclusion of the proteins from the Pd-enriched targeting pathway. The Pd exclusion effect was found to be dominant over the Pd-targeting function of the GPI signal and the Pd-enhancing function of the Pd ectodomain, and it possibly occurs prior to PM localization. Our findings thus uncover a novel Pd-targeting signal and provide, to our knowledge, the first evidence of the cellular mechanism that regulates the sorting of GPI-APs in plants.     

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-d-galactan side chains. The β-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single α-(1→3)-l-arabinofuranosyl residues. Additionally, a substantial proportion of the β-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via β-(1→6)-linkages. The β-(1→6)-galactan side chains are occasionally substituted with α-l-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.Arabinogalactans (AGs) are structurally complex large-branched polysaccharides attached to Hyp residues of many plant cell wall polypeptides. Most proteins glycosylated with AGs (AGPs) have both AG glycosylated domains (glycomodules) and structural or enzymatic domains. However, typical AGPs commonly contain less than 10% protein, suggesting that the AG is the functional part of the molecule (Clarke et al., 1979; Fincher et al., 1983; Kieliszewski and Lamport, 1994; Borner et al., 2003; Xu et al., 2008). Hyp is the most characteristic amino acid present at the glycosylated domain of the AGP, but other amino acids such as Ser, Ala, and Thr are also very common. Type II AG polysaccharides share common structural features based on a β-(1→3)-galactan backbone with β-(1→6)-linked galactan side chains and can be found both on AGPs and rhamnogalacturonan-I (RG-I) pectin (Renard et al., 1991). The galactopyranosyl (Galp) residues can be further substituted with l-arabinofuranosyl (l-Araf) and occasionally also l-rhamnosyl (l-Rha), l-fucosyl (l-Fuc), and glucuronosyl (GlcA; with or without 4-O-methylation) residues (Tsumuraya et al., 1988; Tan et al., 2004; Tryfona et al., 2010). (Sugars mentioned in this work belong to the D-series unless otherwise stated.)The structure of AGs is poorly characterized, and this is mainly due to the great heterogeneity of glycan structures, not only between different AGPs but also even on the same peptide sequence in the same tissue (Estévez et al., 2006). The glycan structure can also be different depending on the developmental stage and tissue type (Tsumuraya et al., 1988), adding to the great heterogeneity of these molecules and therefore limiting their detailed characterization. Molecular and biochemical evidence has indicated that AGPs have specific functions during root formation, promotion of somatic embryogenesis (van Hengel et al., 2002), and attraction of pollen tubes to the style (Cheung et al., 1995). In addition, enhanced secretion efficiency or stability in the cell wall are properties that the AG may confer on the glycosylated protein (Borner et al., 2003). However, it has been difficult to differentiate one species of AGP from another in plant tissues and to assign specific roles to individual AGPs.l-Fuc is present in AGPs in Arabidopsis (Arabidopsis thaliana; van Hengel et al., 2002), radish (Raphanus sativus; Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988), and several other dicot plants such as thyme (Thymus vulgaris; Chun et al., 2001) and celery (Apium graveolens; Lin et al., 2011). Reduction in l-Fuc by 40% in roots of murus1 (mur1) plants resulted in a decrease of 50% in root cell elongation, and eel lectin binding assays suggested that the phenotype was the result of alterations in the composition of root AGPs (van Hengel and Roberts, 2002). An α-(1→2)-fucosyltransferase (FUT) activity for radish primary root AGPs has been described, where an α-l-Araf-(1→3)-β-Galp-(1→6)-Galp trisaccharide was used as exogenous substrate acceptor to mimic an AG polysaccharide in the enzymatic assay (Misawa et al., 1996). Linkage analysis, reactivity with eel lectin, and digestion with α-(1→2)-fucosidase indicated that the l-Fuc residues added are terminal and attached via an α-linkage to the C-2 position of an adjacent l-Araf residue (Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988). Recently, Wu et al. (2010) identified AtFUT4 and AtFUT6 genes encoding FUT proteins specific to AGPs, but the structures of the fucosylated AG generated have not been fully characterized.To gain insights into the synthesis and function of plant AGPs, it would be useful to have mutants altered in their carbohydrate moieties. However, no AG-specific biosynthetic mutants have been characterized, and this, among other reasons, is due to the very limited knowledge of the structure of Arabidopsis AGs (Qu et al., 2008). Moreover, characterization of AG in candidate mutants remains challenging. Even though the structures of some AGs have been proposed using NMR and sugar linkage analyses, the complete structural elucidation of a native AG still remains a formidable task, because NMR spectroscopy and methylation analysis have been largely used to provide information regarding the amount and type of linkages between adjacent glycosyl residues, and AG heterogeneity can confound attempts to build complete structural models. Recently, a modular structure was proposed for AGs on heterologously expressed proteins in tobacco (Nicotiana tabacum; Tan et al., 2010). Tan et al. (2010) proposed that approximately 15-residue repeating blocks of decorated β-(1→3)-trigalactosyl subunits connected by β-(1→6)-linkages were the building blocks of type II AG polysaccharides and concluded that these molecules are far less complex than commonly supposed. Most characterized β-(1→6)-galactan side chains in AGs are reported to be short, of one or two residues (Neukom and Markwalder, 1975; Gane et al., 1995; Gaspar et al., 2001). On the contrary, there are reports of long β-(1→6)-galactan side chains in radish root AGPs (Haque et al., 2005). Similarly, we recently found evidence that wheat (Triticum aestivum) flour endosperm AGP extracts contained long β-(1→6)-galactan side chains heavily substituted with l-Araf at C-3 (Tryfona et al., 2010). This partial structure of the carbohydrate component of wheat flour AGP isolated from water extracts of wheat endosperm was elucidated utilizing a combination of analytical approaches, such as the use of enzymes able to release oligosaccharides specifically from AGs, high-energy matrix-assisted laser desorption ionization (MALDI)-collision-induced dissociation (CID) mass spectrometry (MS), and polysaccharide analysis by carbohydrate gel electrophoresis (PACE; Tryfona et al., 2010). In this work, we applied these techniques to study the carbohydrate component of Arabidopsis leaf AGPs. AG-specific enzyme digestion products were analyzed by PACE and MS, allowing a partial structure to be proposed. We show that endogenous Arabidopsis leaf AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-galactan side chains. These side chains are substituted with l-Araf residues via α-(1→3)-linkages and can vary in length from one up to at least 20 Galp residues. We also found that the β-(1→6)-galactan side chains are substituted mainly with 4-O-methyl-glucuronosyl (4-O-Me-GlcA) at their nonreducing termini, while occasional l-Fuc substitutions were also present via α-(1→2)-linkages on l-Araf residues. In addition, AG oligosaccharides from leaves of the mur1 mutant were identified, and their structures were compared with those isolated from wild-type plants.     

Antiphase Light and Temperature Cycles Affect PHYTOCHROME B-Controlled Ethylene Sensitivity and Biosynthesis,Limiting Leaf Movement and Growth of Arabidopsis

In the natural environment, days are generally warmer than the night, resulting in a positive day/night temperature difference (+DIF). Plants have adapted to these conditions, and when exposed to antiphase light and temperature cycles (cold photoperiod/warm night [−DIF]), most species exhibit reduced elongation growth. To study the physiological mechanism of how light and temperature cycles affect plant growth, we used infrared imaging to dissect growth dynamics under +DIF and −DIF in the model plant Arabidopsis (Arabidopsis thaliana). We found that −DIF altered leaf growth patterns, decreasing the amplitude and delaying the phase of leaf movement. Ethylene application restored leaf growth in −DIF conditions, and constitutive ethylene signaling mutants maintain robust leaf movement amplitudes under −DIF, indicating that ethylene signaling becomes limiting under these conditions. In response to −DIF, the phase of ethylene emission advanced 2 h, but total ethylene emission was not reduced. However, expression analysis on members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ethylene biosynthesis gene family showed that ACS2 activity is specifically suppressed in the petiole region under −DIF conditions. Indeed, petioles of plants under −DIF had reduced ACC content, and application of ACC to the petiole restored leaf growth patterns. Moreover, acs2 mutants displayed reduced leaf movement under +DIF, similar to wild-type plants under −DIF. In addition, we demonstrate that the photoreceptor PHYTOCHROME B restricts ethylene biosynthesis and constrains the −DIF-induced phase shift in rhythmic growth. Our findings provide a mechanistic insight into how fluctuating temperature cycles regulate plant growth.In nature, during the day (light), temperatures are usually higher than during the night (dark). Correspondingly, most plants show optimal growth under such synchronous light and temperature cycles. Increasing the difference between day and night temperature (+DIF) results in increased elongation growth in various species, a phenomenon referred to as thermoperiodism (Went, 1944). The opposite regime, when the temperature of the day (DT) is lower than the temperature of the night (NT), is called −DIF (negative DT/NT difference). Under −DIF conditions, the elongation growth of stems and leaves of various plant species is reduced (Maas and van Hattum, 1998; Carvalho et al., 2002; Thingnaes et al., 2003). Arabidopsis (Arabidopsis thaliana) plants grown under −DIF (DT/NT 12°C/22°C) displayed a reduction in leaf elongation of approximately 20% compared with the control (DT/NT 22°C/12°C; Thingnaes et al., 2003). −DIF is frequently applied in horticulture to produce crops with a desirable compact architecture without the need for growth-retarding chemicals (Myster and Moe, 1995). Despite the economic importance of the application of such temperature regimes in horticulture, the mechanistic basis of the growth reduction under −DIF is still poorly understood.Previously, it was demonstrated that −DIF affects phytohormone signaling in plants. In pea (Pisum sativum), for instance, the −DIF growth reduction correlated with increased catabolism of the phytohormone GA (Stavang et al., 2005). In contrast to pea, active GA levels did not decrease in response to −DIF in Arabidopsis (Thingnaes et al., 2003). On the other hand, the −DIF growth response in Arabidopsis was associated with reduced auxin levels (Thingnaes et al., 2003). The photoreceptor PHYTOCHROME B (PHYB) has been shown to be important for the response to −DIF, as phyB mutants of Arabidopsis (Thingnaes et al., 2008) and cucumber (Cucumis sativus; Patil et al., 2003) are insensitive to −DIF.In this work, the growth-related movement of mature Arabidopsis rosette leaves was analyzed under control (+DIF) and −DIF conditions. Under −DIF, the amplitude of leaf movement was decreased and the phase of movement was later, compared with control plants. The altered leaf growth patterns observed in −DIF could be restored by the application of ethylene. −DIF reduced the expression of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2 (ACS2) in the petiole, which correlated with reduced 1-aminocyclopropane-1-carboxylic acid (ACC) levels and decreased amplitude and delayed phase of leaf movement. Our results indicate that local ACS activity plays an important biological role, despite the fact that ethylene is a gaseous and fast-diffusing hormone. In addition, we demonstrate that in the phyB9 mutant, the phase of leaf movement is almost fully temperature entrained. Finally, ethylene levels and sensitivity are increased in phyB9, suggesting a role for PHYB in constraining temperature-induced shifts in the phase of leaf movement and dampening of leaf movement amplitude by controlling ethylene production and sensitivity.     

Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection

The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCP^O, to an active red form, OCP^R. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCP^R. A comparison of the spectroscopic properties of the RCP with OCP^R indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCP^R form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.     

The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERES.     

Cytoplasmic Nucleation and Atypical Branching Nucleation Generate Endoplasmic Microtubules in Physcomitrella patens

The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.     

Speciation and Distribution of Arsenic in the Nonhyperaccumulator Macrophyte Ceratophyllum demersum

Although arsenic (As) is a common pollutant worldwide, many questions about As metabolism in nonhyperaccumulator plants remain. Concentration- and tissue-dependent speciation and distribution of As was analyzed in the aquatic plant Ceratophyllum demersum to understand As metabolism in nonhyperaccumulator plants. Speciation was analyzed chromatographically (high-performance liquid chromatography-[inductively coupled plasma-mass spectrometry]-[electrospray ionization-mass spectrometry]) in whole-plant extracts and by tissue-resolution confocal x-ray absorption near-edge spectroscopy in intact shock-frozen hydrated leaves, which were also used for analyzing cellular element distribution through x-ray fluorescence. Chromatography revealed up to 20 As-containing species binding more than 60% of accumulated As. Of these, eight were identified as thiol-bound (phytochelatins [PCs], glutathione, and cysteine) species, including three newly identified complexes: Cys-As(III)-PC₂, Cys-As-(GS)₂, and GS-As(III)-desgly-PC₂. Confocal x-ray absorption near-edge spectroscopy showed arsenate, arsenite, As-(GS)₃, and As-PCs with varying ratios in various tissues. The epidermis of mature leaves contained the highest proportion of thiol (mostly PC)-bound As, while in younger leaves, a lower proportion of As was thiol bound. At higher As concentrations, the percentage of unbound arsenite increased in the vein and mesophyll of young mature leaves. At the same time, x-ray fluorescence showed an increase of total As in the vein and mesophyll but not in the epidermis of young mature leaves, while this was reversed for zinc distribution. Thus, As toxicity was correlated with a change in As distribution pattern and As species rather than a general increase in many tissues.Arsenic (As) is ubiquitously present, considered a nonessential metalloid for plants and animals, and poses serious health hazards to humans. High levels of As in soil and drinking water have been reported around the world, with the worst situations in south and southeast Asia, where millions of people are at risk of As poisoning through drinking water and food (Chowdhury et al., 2000; Smedley and Kinniburgh, 2002; Ohno et al., 2007). Recently, As induced yield loss; thus, a threat to the sustainability of food production has been recognized as the other side of the As calamity (Brammer and Ravenscroft, 2009; Panaullah et al., 2009). Considering the enormity of As contamination, phytoremediation or the development of crops that can be grown in contaminated environments without suffering from and accumulating As in edible parts seem to be the most appropriate strategies to counter the detrimental impacts of As. These strategies demand an understanding of the mechanistic details of As uptake, toxicity, and detoxification (Tripathi et al., 2007). The in planta distribution and speciation of As are important aspects in this direction.Inorganic arsenate [HAsO₄²⁻ or As(V)] and arsenite [H₂AsO₃⁻ or As(III)] are the most common forms of As in aquatic and terrestrial environments. Plants take up As(V) through phosphate transporters (Asher and Reay, 1979; Meharg and Macnair, 1990) and As(III) through nodulin26-like intrinsic aquaporins (Isayenkov and Maathuis, 2008; Ma et al., 2008). Inside the cell, As(V) is readily reduced to As(III) through As(V) reductase using reduced glutathione (GSH) as reductant (Duan et al., 2005; Bleeker et al., 2006). As(III) then gets complexed with thiol ligands via GSH and phytochelatins (PCs; Schmöger et al., 2000; Raab et al., 2004). Thus, complexation of As with PCs followed by sequestration of the complex in vacuoles has been suggested as the major mechanism of As detoxification in As-nonhyperaccumulator plants (Bleeker et al., 2006; Song et al., 2010). In contrast, in hyperaccumulator plants, PCs contribute only little (1%–3%) to As complexation (Zhao et al., 2003).An increased synthesis of PCs as well as GSH under As stress has been observed in hypertolerant (Hartley-Whitaker et al., 2001; Bleeker et al., 2003), hyperaccumulator (Zhao et al., 2003; Cai et al., 2004), as well as nonhyperaccumulator (Srivastava et al., 2007; Mishra et al., 2008, 2013) plants. However, the existence of As-PC complexation was concluded only indirectly through individual analysis of As and PCs in fractions after chromatographic separation (Sneller et al., 1999; Schmöger et al., 2000). Raab et al. (2004) for the first time showed complexes of PCs in plant extracts by using HPLC simultaneously coupled with element-specific (inductively coupled plasma-mass spectrometry [ICP-MS]) and molecule-specific (electrospray ionization-mass spectrometry [ESI-MS]) detectors. This method provides information about the diversity of ligands and As species present in plants. However, artifacts during sample preparation, such as ligand exchange for previously weakly bound metal(loids) due to the breakage of cells and subcellular compartments during the extraction of plants, cannot be excluded. In this respect, x-ray absorption spectroscopy of frozen-hydrated tissues has been proven to be a unique technique for the in situ investigation of chemical forms of metal(loids) in biological materials without much prepreparation of samples, thus minimizing the artifacts of sample preparation (Salt et al., 1995; Küpper et al., 2004). X-ray absorption near-edge spectroscopy (XANES) provides speciation of As in tissues typically by fingerprint-like comparison (if quantitative, linear combination fitting) with spectra of appropriate model compounds as standards (Pickering et al., 2000, 2006; Lombi et al., 2002, 2009; Meharg et al., 2008). Furthermore, high-resolution microscopic (µ) x-ray spectroscopy may reveal spatial distribution and speciation of As in intact biological samples (Hokura et al., 2006; Carey et al., 2010, 2011; Kopittke et al., 2012). However, species having almost identical absorption spectra cannot be distinguished by this technique, the use of inappropriate standards for fitting of experimental spectra may lead to misinterpretation, and quantification of minor contributions is always inaccurate in linear combination fitting. Studies regarding the distribution of As have been mostly done in the hyperaccumulator Pteris vittata, showing that the majority of As was accumulated in the pinnae, possibly in vacuoles (Lombi et al., 2002; Hokura et al., 2006; Pickering et al., 2006). In rice (Oryza sativa) grains, it was done with high-resolution techniques such as synchrotron-based microscopic x-ray fluorescence (µ-XRF) and secondary ion mass spectrometry by using either fractured or sectioned dry rice grains (Meharg et al., 2008; Lombi et al., 2009; Moore et al., 2010) or whole fresh rice grains (Carey et al., 2010, 2011). However, little information is available regarding the cellular or subcellular distribution of As in nonhyperaccumulator plants. Moore et al. (2011) investigated the subcellular distribution of As and silicon in rice roots through nanosecondary ion mass spectrometry. Recently, Kopittke et al. (2012) studied the spatial distribution of As in hydrated, fresh roots of cowpea (Vigna unguiculata) using µ-XRF including sequential computed tomography and found differences in As distribution in the plants exposed to As(V) or As(III). However, information about As speciation and distribution in leaf tissues and its relation to toxicity in nonhyperaccumulator plants is still lacking.In this study, we analyzed the speciation (through two complementary techniques) and distribution of As in leaves of the nonhyperaccumulator submerged aquatic plant Ceratophyllum demersum in environmentally relevant conditions. The accumulation and speciation of As were investigated in a concentration-dependent manner at the whole-plant level in fresh extracts through HPLC-(ICP-MS)-(ESI-MS). The in situ speciation at the differential tissue level in the leaf was performed by combining high-resolution µ-XANES and confocal optics on the detector side. Using µ-XRF with full quantification including the correction of x-ray absorption in thick samples, we furthermore investigated changes in the distribution pattern of As, copper (Cu), and zinc (Zn) under sublethal to lethal As exposure in the leaves of C. demersum. To our best knowledge, this is the first report of As speciation in different tissues through confocal µ-XANES and of As distribution in leaves of an As nonhyperaccumulator plant. We also report direct evidence of in situ As-PC complex formation by using µ-XANES spectra of As-(PC₂)₂ and As-PC₃ along with As-(GS)₃ standards and carefully correcting for self-absorption artifacts that distorted µ-x-ray absorption spectroscopy spectra in earlier studies. Since C. demersum has no roots, it takes up all nutrients directly via the leaves, allowing for studies of shoot effects without interference from root uptake and root toxicity. While it is not a crop species, it is still a flowering plant (belonging to the dicotyledons) and has been accepted as a good model for laboratory studies of shoot toxicity in higher plants (Xue et al., 2012). Mechanisms of metal toxicity found in C. demersum are similar to other plants (Küpper et al., 1996), and like other nonaccumulator plants including crops, it detoxifies As by PCs (Mishra et al., 2008). Furthermore, it is widespread in Asia, Europe, and North America, and it has been shown to be good for removing metal from low-concentration waste water (Keskinkan et al., 2004). Additionally, its has also been used successfully in tests of biological life support systems on space flights (Blüm et al., 1994; and many articles about the Aquarack/CEBAS (for Closed Equilibrated Biological Aquatic System) and OMEGAHAB systems). Finally, the structure of C. demersum leaves (round, less than 1 mm diameter) makes this species a suitable model for µ-XRF and µ-XANES measurement. It facilitates the recording of whole-leaf tomograms and tissue-specific µ-XANES without any disturbance such as mixing of intercellular or intracellular fluid while sectioning of the sample, thus avoiding artifacts during sample preparation.