Tryptophan-Independent Indole-3-Acetic Acid Synthesis: Critical Evaluation of the Evidence

Evidence for Trp-independent IAA synthesis is critically reevaluated in the light of tryptophan synthase proteome data, local IAA synthesis and Trp, indole-3-pyruvate, and IAA turnover.Trp-independent synthesis of indole-3-acetic acid (IAA) was proposed back in the early 1990s based on observations from Trp auxotrophs in maize (Zea mays; Wright et al., 1991) and Arabidopsis (Arabidopsis thaliana; Normanly et al., 1993). Recently, Wang et al. (2015) published new data suggesting that a cytosolic indole synthase (INS) may catalyze the first step separating the Trp-dependent and Trp-independent pathways in Arabidopsis. If this is the case, it would be a major breakthrough; however, in this article, I critically evaluate both recent and older evidence for the Trp-independent route and suggest that the INS is more likely to participate in Trp-dependent IAA production.The original work supporting Trp-independent IAA production was carried out prior to the availability of genome/proteome data and before the discovery that the final step of Trp-dependent IAA synthesis is carried out by a large number of YUCCA homologs operating in a highly localized manner (Zhao, 2008). I argue that experimental data supporting the Trp-independent route needs to be reconsidered in light of complete proteome data. Further, the evidence from feeding labeled compounds should be critically evaluated in light of recent data on the highly localized nature of IAA synthesis as well as older quantitative data on Trp, indole-3-pyruvic acid (IPA), and IAA turnover from my own laboratory (Cooney and Nonhebel, 1991). I conclude that evidence for the Trp-independent route is at best equivocal, and that it is not a conserved source of IAA in angiosperms.Figure 1 shows the major Trp-dependent route for IAA production whereby Trp, produced by the concerted action of Trp synthase α- and β-subunits, is converted to IAA in a further two steps catalyzed by Trp aminotransferase and the flavin monooxygenases commonly known as YUCCA (Mashiguchi et al., 2011; Won et al., 2011). This is compared with the Trp-independent route in which IAA may be produced from free indole by an unknown route (Ouyang et al., 2000; Wang et al., 2015).Open in a separate windowFigure 1.Outline of the major pathway for Trp-dependent IAA synthesis and the proposed Trp-independent route. The role proposed for Trp synthase beta (TSB) homologs discussed in the present paper is also shown. For clarity, reactions are simplified to show only the major compounds relevant to IAA synthesis.The Trp-independent route was originally based on data from Trp auxotrophs that have mutations in genes encoding either the α- or β-subunits of Trp synthase. The α-subunit catalyzes the removal of the side chain from indole-3-glycerol phosphate, passing the indole product directly to the β-subunit where the Trp side chain is created from a Ser substrate (Pan et al., 1997). In plants, this is a chloroplast-localized enzyme. Elevated levels of IAA have been reported in Trp auxotrophs of both maize and Arabidopsis. However, the trp3-1 and trp2-1 mutants of Arabidopsis, deficient in the α- and β-subunits, respectively, only showed an increase in total IAA measured following conjugate hydrolysis. No difference in free IAA levels was found (Normanly et al., 1993). The orange pericarp (orp) maize mutant was reported to have 50 times more IAA than the wild type (Wright et al., 1991). However, this was also total IAA; no data on free IAA were published. Work by Müller and Weiler (2000) indicated that IAA measured following conjugate hydrolysis could have originated via the degradation of indole-3-glycerol phosphate that accumulates in trp3-1 mutants. Further doubt regarding the accuracy of IAA measurements following conjugate hydrolysis has recently been published. Yu et al. (2015) have shown that conjugate hydrolysis treatment substantially overestimates the actual conjugated IAA due to degradation of glucobrassicin and proteins. In addition, neither report (Wright et al., 1991; Normanly et al., 1993) described a high auxin phenotype for the Trp auxotrophs. This contrasts with the superroot1 (sur1) and sur2 mutants, where the accumulation of indole intermediates resulted in a high level of free IAA as well as a high auxin phenotype (Boerjan et al., 1995; Delarue et al., 1998). It is therefore doubtful that Trp auxotrophs actually accumulate more IAA than the wild-type plants.In addition, proteome data have revealed new homologs of TSB in both Arabidopsis and maize that may contribute to Trp production in TSB mutants; these have not been considered in arguments supporting Trp-independent IAA synthesis. Maize orp has mutations in two TSB genes, resulting in a seedling lethal phenotype with high levels of accumulated indole. However, proteome sequence information now indicates that maize has three TSB genes. Plants and bacteria have divergent forms of TSB, type 1 and type 2 (Xie et al., 2001); the major TSB genes responsible for Trp synthase activity in maize and Arabidopsis are type 1. The third maize TSB gene, maize locus ID GRMZM2G054465, is a member of the TSB type 2 group. Its product is reported not to interact directly with a Trp synthase alpha (TSA) subunit but has experimentally demonstrated catalytic activity converting indole and Ser to Trp (Yin et al., 2010). This type 2 TSB may allow orp plants to make sufficient Trp for IAA production from the accumulated free indole.When the original work on trp2 mutants of Arabidopsis was carried out, two TSB genes were known (Last et al., 1991). As the trp2 plants were deficient only in TSB1, they were able to make sufficient Trp to survive under low-light conditions. Full proteome data now indicate that Arabidopsis has four TSB-like genes; in addition to TSB1 and TSB2, there is a third type 1 TSB gene, Arabidopsis locus ID AT5G28237. The product of this gene has not been experimentally characterized. The fourth gene, AT5G38530, encodes a type 2 TSB with demonstrated catalytic activity similar to ZmTSB type 2 mentioned above (Yin et al., 2010). Thus, the trp2 plants may also make enough Trp for IAA production. It is even possible that one of the minor forms of TSB has a specific role in IAA production. Type 2 TSBs are conserved throughout the plant kingdom, and the biological role for this protein is not known (Xie et al., 2001).A phylogenetic analysis of type 1 TSBs is shown in Figure 2. This indicates that the product of AT5G28237 belongs to a eudicot-conserved TSB type 1-like clade, divergent from that containing major experimentally characterized TSBs. A multiple sequence alignment (not shown) reveals that members of this divergent clade have a shortened N terminus with respect to the major chloroplast-localized TSB proteins. A localization prediction carried out in CELLO (Yu et al., 2006) suggests a cytosolic location for these proteins. Examination of EST databases indicates that the genes encoding these proteins are expressed. It is possible that the product of AT5G28237 could interact with the cytosolic INS studied by Wang et al. (2015), or separately with its indole product, to produce Trp that is further converted to IAA.Open in a separate windowFigure 2.Phylogeny of TSB type 1 homologs from Oryza sativa (LOC_Os), Sorghum bicolor (Sobic), Z. mays (GRMZM), Arabidopsis (AT), Brassica rapa (Brara), Solanum lycopersicum (Solyc), Populus trichocarpa (Potri), and Physcomitrella patens (Phpat). Protein sequences were downloaded from Phytozome v10.2 (Goodstein et al., 2012). The phylogenetic analysis was conducted in MEGA6 (http://megasoftware.net; Tamura et al., 2013) with multiple sequence alignment by MUSCLE (Edgar, 2004) and evolutionary history inferred using the neighbor-joining method (Saitou and Nei, 1987). The optimal tree is shown; the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale; the scale bar indicates the number of amino acid substitutions per site. It is rooted with type 1 TSBs from the moss P. patens.The second major line of evidence for Trp-independent IAA synthesis comes from isotopic labeling experiments. Wright et al. (1991) observed greater incorporation of ²H into IAA than Trp in orp seedlings grown on ²H₂O. Normanly et al. (1993) reported higher enrichment of ¹⁵N in IAA than Trp in trp2-1 mutants of Arabidopsis grown on ¹⁵N anthranilate; very poor incorporation of deuterium from ²H-Trp into IAA was reported in the trp2-1 plants. A number of similar reports relating to other plants have been published showing differences in the incorporation of label from Trp into IAA depending on experimental tissue and environmental conditions (e.g. Michalczuk et al., 1992; Rapparini et al., 2002; Sztein et al., 2002). This evidence has been persuasive; however, it assumes a single pool of Trp to which ¹⁵N anthranilate and ²H-Trp contribute and from which IAA is made. If Trp is made at different rates in different parts of the plant, and/or exogenous ²H-Trp does not equilibrate with newly synthesized Trp, then the ratio of ¹⁵N to ²H in Trp will vary in different plant organs/tissues/cells. Trp turnover and thus incorporation of label from ¹⁵N anthranilate are likely to differ substantially throughout the plant, with the highest rates of labeling occurring in cells with high rates of protein synthesis. This would not be a problem for the experiment if IAA is made at equal rates in different parts of the plant, but we know it is not. The Trp aminotransferase/YUCCA pathway of IAA synthesis elegantly shown to be responsible for the bulk of IAA synthesis (Mashiguchi et al., 2011; Won et al., 2011) appears to be locally controlled in Arabidopsis via 11 different YUCCA-encoding genes that have highly localized expression (Zhao, 2008). Adding to the complication is the need for ¹⁵N anthranilate and ²H-Trp to move into and through the plant to regions of Trp and IAA synthesis, respectively. This is likely to occur at different rates due to differing transporter requirements.Data from my own laboratory (Cooney and Nonhebel, 1991) is particularly relevant to this discussion. We monitored incorporation of ²H from deuterated water into IAA and Trp in tomato (S. lycopersicum) shoots. Unlike the other studies, we also measured the incorporation of label into IPA. Our data showed that IPA became labeled at a rate consistent with this compound acting as the major/sole precursor of IAA. Crucially, the proportion of labeled Trp was lower than ²H-IPA. Our interpretation of these data was that IPA and IAA were produced from newly synthesized Trp, and that Trp was not uniformly labeled throughout the shoot. At the time, we suggested different subcellular pools of Trp; this may be the case, but in light of new knowledge of localized IAA synthesis, it is most likely that substantial differences in Trp and IAA turnover in different cells/tissues may be the reason for these observations.The arguments above cast doubt on the existence of the Trp-independent route; however, a recent publication by Wang et al. (2015) claims to provide new evidence for its importance. They present the interesting finding that Arabidopsis plants with a null mutation in INS, a cytosolic TSA homolog previously shown to have indole-3-glycerol phosphate lyase (IGL) activity (Zhang et al., 2008), had reduced levels of IAA. The mutation particularly affected early embryo development. I suggest that the INS may make a contribution to IAA synthesis, but the only specific evidence that it does so via a Trp-independent route is the observation that the ins-1 mutation has an additive effect with the weakly ethylene insensitive8-1 Trp aminotransferase mutation. This evidence is indicative rather than conclusive. The possibility that INS may act in concert with a minor TSB homolog, as suggested in Figure 1, needs to be considered.In addition, Wang et al. (2015) focus on Arabidopsis alone. If INS has a key role in IAA synthesis, then evolutionary theory predicts a conserved protein with wide taxonomic distribution. On the contrary, an exhaustive BLAST search (Altschul et al., 1997) of diverse taxa in Phytozome v10.2 (Goodstein et al., 2012) and GenBank (Benson et al., 2013) revealed that INS orthologs with cytosolic prediction and shortened N terminus occur only in members of the Brassicaceae (Eutrema salsugineum, Arabis alpina, Camelina sativa, Capsella rubella, Brassica napus, Boechera stricta, Arabidopsis lyrata, B. rapa) and in Tarenaya hassleriana from the Brassicaceae sister family, the Cleomaceae. The phylogenetic tree in Figure 3 shows relationships between INS and TSA homologs from several plant species and indicates the separate clade of cytosolic INS homologs in the Brassicaceae. In this diagram, the sequence most closely related to INS from another group is that from tomato. This protein is the only TSA found in tomato and has an unambiguous chloroplast signal peptide. P. trichocarpa and M. truncatula as well as other eudicots outside the Brassicaeae and Cleomaceae also lack cytosolic TSA homologs. Furthermore, INS and its orthologs are phylogenetically distinct from the other experimentally characterized indole-3-glycerol phosphate lyases benzoxazin1 and IGL (Frey et al., 2000) and their orthologs. The latter are restricted to the grasses where they are involved in the production of cyclic hydroxamic acid defense compounds (Frey et al., 2000). The grasses also have an additional separate clade of cytosolic TSA homologs, although work by Kriechbaumer et al. (2008) did not detect any catalytic activity for the product of GRMZM2G046191_T01. The phylogeny of INS and its orthologs would suggest the major role of these proteins may be the production of lineage-specific metabolites such as the indole-derived defense compounds produced in grasses; any role in IAA synthesis may be incidental and restricted to the Brassicaeae and Cleomaceae.Open in a separate windowFigure 3.Phylogeny of TSA homologs from O. sativa (LOC_Os), S. bicolor (Sobic), Z. mays (GRMZM), Arabidopsis (AT), A. lyrata (Alyrata), B. rapa (Brara), S. lycopersicum (Solyc), Medicago truncatula (Medtr), P. trichocarpa (Potri), and P. patens (Phpat). Protein sequences were downloaded from Phytozome v10.2 (Goodstein et al., 2012). The phylogenetic analysis was conducted in MEGA6 (Tamura et al., 2013) with multiple sequence alignment by MUSCLE (Edgar, 2004) and evolutionary history inferred using the neighbor-joining method (Saitou and Nei, 1987). The rooted optimal tree is shown; the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale; the scale bar indicates the number of amino acid substitutions per site. It is rooted with the TSA ortholog from the moss P. patens.In conclusion, I contend that experimental data relating to IAA synthesis in Arabidopsis, including that suggesting the involvement of a cytosolic INS, can be explained by the Trp-dependent IAA synthesis pathway. I show that INS and its orthologs are not found outside the Brassicaceae and a closely related sister clade; any alternative IAA synthesis pathway in which they may be involved is likely to have similar limited taxonomic occurrence. Furthermore, Arabidopsis and its relatives contain two additional TSB homologs that could convert free indole into Trp. Curiously, both of these proteins have a wider taxonomic distribution. A priority for further experimental work should be testing the involvement of minor TSB homologs in IAA synthesis, including the highly conserved type 2 TSBs as well as a eudicot-specific clade of possibly cytosolic type 1 TSBs. Work would also have to establish whether free indole exists in plants other than the Brassicaceae and the grasses. Finally, I argue that isotope-labeling experiments do not provide strong support for the Trp-independent route, as IAA production is highly localized. Previously published data from my laboratory clearly show that the main Trp-dependent IAA precursor IPA becomes more highly labeled from ²H₂O than Trp, even though the latter is produced from Trp in a single reaction. Thus, it cannot be argued that differences in isotope enrichment between Trp and IAA demonstrate the existence of a Trp-independent route.     

Impaired Auxin Biosynthesis in the defective endosperm18 Mutant Is Due to Mutational Loss of Expression in the ZmYuc1 Gene Encoding Endosperm-Specific YUCCA1 Protein in Maize

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.The phytohormone auxin, as a signaling molecule, controls and coordinates numerous aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most predominant in planta auxin and regulates diverse processes, including cell division, cell elongation, formation and maintenance of meristems, vascular tissue differentiation, phototropism, flowering, and endosperm and embryo growth in developing seeds (Davies, 2010). Despite its critical roles, basic components of IAA biosynthesis are poorly understood, compared with transport and signaling aspects. However, the use of appropriate genetic screens in Arabidopsis (Arabidopsis thaliana) and the use of sensitive analytical tools in the identification of metabolic intermediates have led to significant advancements toward a better understanding of biosynthesis. Currently, there are four proposed Trp-dependent pathways of de novo IAA biosynthesis in plants (Woodward and Bartel, 2005; Pollmann et al., 2009; Normanly, 2010); of these, indole-3-pyruvic acid (IPA) was recently suggested to predominate in Arabidopsis (Mashiguchi et al., 2011; Won et al., 2011; Stepanova et al., 2011) and in pea (Pisum sativum) seeds (Tivendale et al., 2012).The first step of the IPA pathway involves the conversion of Trp to IPA by Trp aminotransferases, first demonstrated in Arabidopsis by Stepanova et al. (2008) and Tao et al. (2008). The mutants of Arabidopsis Trp-aminotransferase (taa1) are defective in shade avoidance syndrome due to reduced levels of IAA. In maize (Zea mays), orthologs of the TAA1 gene include an endosperm-specific gene, ZmTar1 (for TA-Related1; Chourey et al., 2010) and Vanishing tassel2 (Vt2), which encode grass-specific Trp aminotransferases (Phillips et al., 2011). The vt2 mutant is marked by severe developmental abnormality, attributed to approximately 60% reduced IAA levels in the mutant seedlings. These results are significant in showing the functionality of the TAR enzyme and the IPA pathway in IAA biosynthesis in maize. Recently, it was suggested that the IPA pathway also involves the YUCCA (YUC) genes, which encode flavin monooxygenases that are now believed to catalyze the conversion of IPA to IAA (Phillips et al., 2011; Mashiguchi et al., 2011; Stepanova et al., 2011; Won et al., 2011; Kriechbaumer et al., 2012). This is based in part on evidence that the Arabidopsis YUC2 protein, expressed in Escherichia coli, converted IPA to IAA in vitro (Mashiguchi et al., 2011). In Arabidopsis, three Yuc genes, Yuc-1, -4, and -10, are expressed in an overlapping fashion in developing seeds and are considered essential in embryogenesis (Cheng et al., 2007); however, single or double mutant yuc1 yuc4 do not show detectable defects in embryogenesis or seed phenotype.Orthologs of the AtYuc genes are now described in several plant groups, including maize (Gallavotti et al., 2008; LeClere et al., 2010). The first Yuc-like gene in maize was isolated through positional cloning of the sparse inflorescence1 (spi1) locus; spi1 mutants showed auxin-deficient-related characteristics in the male inflorescence (Gallavotti et al., 2008). The second gene, ZmYuc1, is highly endosperm specific and its temporal expression pattern coincided with IAA biosynthesis at various stages of seed development (LeClere et al., 2010). In pea, two highly similar PsYuc-like genes, PsYuc1 and PsYuc2, showed seed- and root-specific expression, respectively (Tivendale et al., 2010). Metabolic studies in pea, however, showed that only the roots but not seeds can metabolize Trp to IAA through the proposed TAM pathway (Quittenden et al., 2009; Tivendale et al., 2010).In contrast with many studies on auxin-related mutants that affect vegetative parts of the plant, very limited data are available on auxin mutants affecting seed development, even though seeds accumulate higher levels of IAA than any other tissue of the plant. In maize, endosperm synthesizes nearly 100- to 500-fold higher levels of IAA relative to vegetative tissues (Jensen and Bandurski, 1994; LeClere et al., 2008; Phillips et al., 2011). The significance of the large abundance of IAA in developing endosperm remains to be understood, except that it may be used during the very early stages of seed germination because >90% of the total IAA is in biologically inactive conjugated storage form (Jensen and Bandurski, 1994; LeClere et al., 2008). Such a role in germination is consistent with the fact that there are very few viable seed mutants reported in maize that are linked to IAA deficiency, although single-locus recessive mutants (defective kernels [dek]) with various abnormalities in either embryo or endosperm development and with low IAA levels (measured by ELISA) were reported by Lur and Setter (1993). It is significant in this regard that a viable defective endosperm-B18 (hereafter, de18) was identified as associated with IAA deficiency (Torti et al., 1986). Although not quantified by mass spectrometry, de18 endosperms contained total IAA levels (including conjugates) in the range of 6% to 0.3% of the wild type B37 (hereafter, De18) values, during 12 to 40 d after pollination (DAP). At the early stages, the mutant seed phenotype is <50% of the wild type in seed weight, and throughout seed development, mutant seeds are reduced in kernel size and accumulate less dry matter. Furthermore, application of the synthetic auxin, naphthalene acetic acid, to developing seeds largely rescued the de18 mutant phenotype, indicating impairment in IAA biosynthesis or metabolism as the cause of the phenotypic changes (Torti et al., 1986). Recent cellular-level studies also indicated the IAA deficiency of the de18 endosperm; high levels of immunosignal for IAA were detected in the basal endosperm transfer layer (BETL), aleurone, embryo surrounding region domains, and maternal chalazal tissue in De18 but not in the mutant (Forestan et al., 2010). Overall, the maize de18 and the pea tar2 (Tivendale et al., 2012) mutants are thus far the only seed-specific viable mutants linked to auxin deficiency. The objective of this study is to further extend our knowledge on IAA deficit in the de18 kernels, to specifically analyze temporal expression of two major IAA biosynthetic genes and to elucidate the possible molecular basis of the mutant. Our collective data, based on the cloning and sequencing of ZmYuc1 and on mapping studies, indicate that ZmYuc1 and De18 are tightly associated and that the aberrant YUC1 protein in de18 is the causal basis of IAA deficiency and the small seed phenotype in that mutant.     

Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH

CATION EXCHANGERs CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3 in regulating apoplastic pH and describe how they contribute to auxin transport using the guard cell’s response as readout of hormone signaling and cross talk. We show that indole-3-acetic acid (IAA) inhibition of abscisic acid (ABA)-induced stomatal closure is impaired in cax1, cax3, and cax1/cax3. These mutants exhibited constitutive hypopolarization of the plasma membrane, and time-course analyses of membrane potential revealed that IAA-induced hyperpolarization of the plasma membrane is also altered in these mutants. Both ethylene and 1-naphthalene acetic acid inhibited ABA-triggered stomatal closure in cax1, cax3, and cax1/cax3, suggesting that auxin signaling cascades were functional and that a defect in IAA transport caused the phenotype of the cax mutants. Consistent with this finding, chemical inhibition of AUX1 in wild-type plants phenocopied the cax mutants. We also found that cax1/cax3 mutants have a higher apoplastic pH than the wild type, further supporting the hypothesis that there is a defect in IAA import in the cax mutants. Accordingly, we were able to fully restore IAA inhibition of ABA-induced stomatal closure in cax1, cax3, and cax1/cax3 when stomatal movement assays were carried out at a lower extracellular pH. Our results suggest a network linking the vacuolar cation exchangers to apoplastic pH maintenance that plays a crucial role in cellular processes.Stomata are pores at the surface of the leaves, gating water loss and gas exchange between plants and the atmosphere. One stoma is formed by two specialized guard cells that are able to modulate their size and shape to control stomatal aperture in response to various signals, including water status, hormonal stimuli, CO₂ levels, light, or temperature (Kwak et al., 2008). These stomatal movements are regulated by ion fluxes in guard cells, the changes in the osmoticum status being compensated by water movement, which modifies the cell’s volume. Ion transport between the cell and ion stores (vacuole, apoplastic space) must be therefore tightly controlled, and any change in the guard cell’s ability to regulate this can compromise its faculty to trigger stomatal movement.Calcium ion (Ca²⁺) is one ion that regulates stomatal movements, and its cytosolic concentration is controlled by both influx, via plasma membrane channels, and release from internal stores such as vacuoles and the endoplasmic reticulum. Calcium transport from the vacuole is ensured, at least in part, by members of the Cation Exchanger (CAX) family (Punshon et al., 2012). Six members of this family are found in Arabidopsis (Arabidopsis thaliana); all use a proton gradient generated by the vacuolar H⁺-ATPase (VHA) or the vacuolar pyrophosphatase (AVP1) to energize their activity. CAX1 and CAX3 are the closest homologs within the family and have been proposed to play similar roles in Ca²⁺ homeostasis (Zhao et al., 2008). However, biochemical characterization highlighted differences in their respective rates of Ca²⁺ transport, and they have been proposed to function as heterodimers, with unique properties associated with this structure (Cheng et al., 2005).Among common phenotypes of cax1 and cax3, an increased sensitivity to abscisic acid (ABA; Zhao et al., 2008) suggests a function for these transporters in modulating hormone signaling. ABA is well known for its role in triggering stomatal closure, whereas auxin, ethylene, or cytokinins can counteract its effect. Auxin in particular is also essential in governing plant development, including root architecture, tropisms and polarity, apical dominance, tissue differentiation, and plant development. Tight control of its distribution throughout the plant is achieved via ubiquitous and specific expression of members of three transporter families, acting together in mediating indole-3-acetic acid (IAA) fluxes (Krecek et al., 2009).The unique pattern of auxin distribution is predominately due to the asymmetrical localization of members of the PIN-FORMED (PIN) family of auxin exporters (Zazímalová et al., 2010). In Arabidopsis, this family comprises eight members, whose spatiotemporal expression is responsible for the auxin gradient observed in many plant tissues (Paponov et al., 2005). In addition, most members of the ATP-binding cassette (ABC)-type family of exporter ABCB (ABCB/multidrug resistance/phosphoglycoprotein) have been shown to mediate auxin export from the cell (Geisler and Murphy, 2006). Auxin import is mainly ensured by (1) active transport of IAA by members of the AUX1/LAX family proteins (Geisler and Murphy, 2006), and (2) passive diffusion across the plasma membrane. AUX1 activity was demonstrated to be pH-dependent (Yang et al., 2006), IAA transport being optimal at acidic pH (5.5–6), and dramatically reduced at higher values. It is interesting that passive, pH-dependent IAA diffusion across the plasma membrane also accounts for an important part of IAA transport and signaling. At apoplastic pH (5.5), between 10% and 25% of IAA is protonated (Yang et al., 2006), which allows for free diffusion of IAA through the membrane. In contrast, the ratio between protonated and deprotonated IAA (IAAH/IAA⁻) falls to 1% to 5% when pH exceeds 6.5, preventing it from being passively transported into the cytoplasm (Yang et al., 2006). These two aspects make control of the apoplastic pH crucial in the regulation of auxin signaling, as it modulates all the known routes of IAA import. Such a tight pH constraint is ensured by plasma membrane-localized Arabidopsis H+-ATPases (AHA; Haruta et al., 2010) that transport protons from the cytosol to the extracellular space.Our work presents the characterization of two vacuolar transporters’ abilities to modulate the apoplastic pH, and therefore contribute to proper auxin transport and signaling. Our results highlight the effects of mutations in CAX1 and CAX3 in plant development and in stomatal functioning, providing new insights for understanding hormone signaling in plants as well as plant adaptation to stress conditions via hormone cross talk.     

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.     

Auxin Produced by the Indole-3-Pyruvic Acid Pathway Regulates Development and Gemmae Dormancy in the Liverwort Marchantia polymorpha

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss- and gain-of-function alleles for auxin synthesis- and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

The native auxin, indole-3-acetic acid (IAA), is a major regulator of plant growth and development. Its nonuniform distribution between cells and tissues underlies the spatiotemporal coordination of many developmental events and responses to environmental stimuli. The regulation of auxin gradients and the formation of auxin maxima/minima most likely involve the regulation of both metabolic and transport processes. In this article, we have demonstrated that 2-oxindole-3-acetic acid (oxIAA) is a major primary IAA catabolite formed in Arabidopsis thaliana root tissues. OxIAA had little biological activity and was formed rapidly and irreversibly in response to increases in auxin levels. We further showed that there is cell type–specific regulation of oxIAA levels in the Arabidopsis root apex. We propose that oxIAA is an important element in the regulation of output from auxin gradients and, therefore, in the regulation of auxin homeostasis and response mechanisms.     

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.     

Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [¹⁴C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.Plant oils are an important natural resource to meet the increasing demands of food, feed, biofuel, and industrial applications (Lu et al., 2011; Snapp and Lu, 2012). The fatty acid composition in the triacylglycerols (TAG), especially the contents of polyunsaturated fatty acids (PUFA) or other specialized structures, such as hydroxy, epoxy, or conjugated groups, determines the properties and thus the uses of plant oils (Dyer and Mullen, 2008; Dyer et al., 2008; Pinzi et al., 2009; Riediger et al., 2009). To effectively modify seed oils tailored for different uses, it is necessary to understand the fundamental aspects of how plant fatty acids are synthesized and accumulated in seed oils.In developing oilseeds, fatty acids are synthesized in plastids and are exported into the cytosol mainly as oleic acid, 18:1 (carbon number:double bonds), and a small amount of palmitic acid (16:0) and stearic acid (18:0; Ohlrogge and Browse, 1995). Further modification of 18:1 occurs on the endoplasmic reticulum in two major pathways (Fig. 1): (1) the 18:1-CoA may be elongated into 20:1- to 22:1-CoA esters by a fatty acid elongase, FAE1 (Kunst et al., 1992); (2) the dominant flux of 18:1 in many oilseeds is to enter the membrane lipid phosphatidylcholine (PC; Shanklin and Cahoon, 1998; Bates and Browse, 2012), where they can be desaturated by the endoplasmic reticulum-localized fatty acid desaturases including the oleate desaturase, FAD2, and the linoleate desaturase, FAD3, to produce the polyunsaturated linoleic acid (18:2) and α-linolenic acid (18:3; Browse et al., 1993; Okuley et al., 1994). The PUFA may be removed from PC to enter the acyl-CoA pool, or PUFA-rich diacylglycerol (DAG) may be derived from PC by removal of the phosphocholine headgroup (Bates and Browse, 2012). The PUFA-rich TAG are then produced from de novo-synthesized DAG or PC-derived DAG (Bates and Browse, 2012) and PUFA-CoA by the acyl-CoA:diacylglycerol acyltransferases (DGAT; Hobbs et al., 1999; Zou et al., 1999). Alternatively, PUFA may be directly transferred from PC onto DAG to form TAG by an acyl-CoA-independent phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). Recent results demonstrated that DGAT and PDAT are responsible for the majority of TAG synthesized in Arabidopsis (Arabidopsis thaliana) seeds (Zhang et al., 2009).Open in a separate windowFigure 1.Reactions involved in the flux of fatty acids into TAG. De novo glycerolipid synthesis is shown in white arrows, acyl transfer reactions are indicated by dashed lines, and the movement of the lipid glycerol backbone through the pathway is shown in solid lines. Major reactions (in thick lines) controlling the flux of fatty acid from PC into TAG are as follows: LPC acylation reaction of acyl editing by LPCAT (A); PC deacylation reaction of acyl editing by the reverse action of LPCAT or phospholipase A (B); and the interconversion of DAG and PC by PDCT (C). Substrates are in boldface, enzymatic reactions are in italics. FAD, Fatty acid desaturase; FAS, fatty acid synthase; GPAT, acyl-CoA:G3P acyltransferase; LPA, lysophosphatidic acid; LPAT, acyl-CoA:LPA acyltransferase; PA, phosphatidic acid; PLC, phospholipase C; PLD, phospholipase D.The above TAG synthesis model highlights the importance of acyl fluxes through PC for PUFA enrichment in plant oils. However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the enzymes/genes that control these fluxes known. The traditional view is that 18:1 enters PC through de novo glycerolipid synthesis (Fig. 1; Kennedy, 1961): the sequential acylation of glycerol-3-phosphate (G3P) at the sn-1 and sn-2 positions produces phosphatidic acid; subsequent removal of the phosphate group at the sn-3 position of phosphatidic acid by phosphatidic acid phosphatases (PAPs) produces de novo DAG; finally, PC is formed from DAG by a cytidine-5′-diphosphocholine:diacylglycerol cholinephosphotransferase (CPT; Slack et al., 1983; Goode and Dewey, 1999). However, metabolic labeling experiments in many different plant tissues by us and others (Williams et al., 2000; Bates et al., 2007, 2009; Bates and Browse, 2012; Tjellström et al., 2012) have demonstrated that the majority of newly synthesized fatty acids (e.g. 18:1) enter PC by a process termed “acyl editing” rather than by proceeding through de novo PC synthesis. Acyl editing is a deacylation-reacylation cycle of PC that exchanges the fatty acids on PC with fatty acids in the acyl-CoA pool (Fig. 1, A and B). Through acyl editing, newly synthesized 18:1 can be incorporated into PC for desaturation and PUFA can be released from PC to the acyl-CoA pool to be utilized for glycerolipid synthesis.Additionally, there is accumulating evidence that many plants utilize PC-derived DAG to synthesize TAG laden with PUFA (Bates and Browse, 2012). PC-derived DAG may be synthesized through the reverse reaction of the CPT (Slack et al., 1983, 1985) or by the phospholipases C and D (followed by PAP). However, our recent discovery indicates that the main PC-to-DAG conversion is catalyzed by a phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through the phosphocholine headgroup exchange between PC and DAG (Fig. 1C; Lu et al., 2009; Hu et al., 2012). The PDCT is encoded by the REDUCED OLEATE DESATURATION1 (ROD1) gene (At3g15820) in Arabidopsis, which is responsible for about 40% of the flux of PUFA from PC through DAG into TAG synthesis (Lu et al., 2009). Acyl editing and PC-DAG interconversion through PDCT may work together to generate PUFA-rich TAG in oilseed plants (Bates and Browse, 2012).The enzymes/genes involved in the incorporation of 18:1 into PC through acyl editing are not known. However, stereochemical localization of newly synthesized fatty acid incorporation into PC predominantly at the sn-2 position (Bates et al., 2007, 2009; Tjellström et al., 2012) strongly suggest that the acyl editing cycle proceeds through the acylation of lysophosphatidylcholine (LPC) by acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs [Enzyme Commission 2.3.1.23]; Fig. 1A). High LPCAT activity has been detected in many different oilseed plants that accumulate large amounts of PUFA in TAG (Stymne and Stobart, 1987; Bates and Browse, 2012), suggesting the potential ubiquitous involvement of LPCAT in the generation of PUFA-rich TAG. Several possible pathways for the removal of acyl groups from PC to generate the lysophosphatidylcholine within the acyl editing cycle have been proposed. The acyl groups may be released from PC to enter the acyl-CoA pool via the reverse reactions of LPCATs (Stymne and Stobart, 1984) or by reactions of phospholipase A (Chen et al., 2011) followed by the acyl-CoA synthetases (Shockey et al., 2002). The main focus of this study was to identify the genes and enzymes involved in the incorporation of fatty acids into PC through acyl editing in Arabidopsis and to quantify the contribution of acyl editing and PDCT-based PC-DAG interconversion to controlling the flux of PUFA from PC into TAG. Herein, we demonstrate that mutants of two Arabidopsis genes encoding LPCATs (At1g12640 [LPCAT1] and At1g63050 [LPCAT2]) have reduced TAG PUFA content. Analysis of the acyl-editing cycle through metabolic labeling of developing seeds with [¹⁴C]acetate indicate that the lpcat1/lpcat2 double mutant was devoid of acyl editing-based incorporation of newly synthesized fatty acids into PC, indicating that these two genes are responsible for the acylation of LPC during acyl editing. Additionally, the triple mutant rod1/lpcat1/lpcat2 indicated that PDCT-based PC-DAG interconversion and acyl editing together provide two-thirds of the flux of PUFA from PC to TAG in Arabidopsis seeds.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii

Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.     

Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation

Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.Modified fatty acids (mFAs; sometimes referred to as unusual fatty acids) obtained from plants play important roles in industrial applications as lubricants, protective coatings, plastics, inks, cosmetics, etc. The hundreds of potential industrial uses of mFAs have led to considerable interest in exploring their production in transgenic crop plants. mFAs are produced by a limited number of species, and the transfer of genes encoding mFA-producing enzymes from source plants to heterologous hosts has generally resulted in only modest accumulation, usually less than 20% of the desired mFA in transgenic seed (Napier, 2007) compared with levels found in the natural source. For example, ricinoleic acid accounts for more than 90% of the fatty acid of castor bean (Ricinus communis) seeds, and tung (Aleuites fordii) seeds accumulate more than 80% α-eleostearic acid (Thelen and Ohlrogge, 2002; Drexler et al., 2003). In order to elevate the content of mFAs in the engineered plants to that found in the native plant, it is necessary to (1) optimize the synthesis of mFA (Mekhedov et al., 2001), (2) minimize its degradation (Eccleston and Ohlrogge, 1998), and (3) optimize its incorporation into triacylglycerol (TAG; Bafor et al., 1990; Bates and Browse, 2011; van Erp et al., 2011).Cyclic fatty acids (CFAs) are desirable for numerous industrial applications. The strained bond angles of the carbocyclic ring contribute to their unique chemistry and physical properties, and hydrogenation of CFAs results in ring opening to produce methyl-branched fatty acids. Branched chain fatty acids are ideally suited for the oleochemical industry as feedstocks for the production of lubricants, plastics, paints, dyes, and coatings (Carlsson et al., 2011). Cyclopropane fatty acids (CPAs) have been found in certain gymnosperms, Malvales, Litchi spp., and other Sapindales species. They accumulate to as much as 40% in seeds of Litchi chinensis (Vickery, 1980; Gaydou et al., 1993). Sterculia foetida accumulates the desaturated CFA (i.e. cyclopropene fatty acid) to more than 60% of its seed oil (Bohannon and Kleiman, 1978; Pasha and Ahmad, 1992). The first step in its synthesis is the formation of the CPA by the cyclopropane synthase (CPS) enzyme, which transfers a methyl group to C9 of the oleoyl-phospholipid followed by cyclization to form the cyclopropane ring (Grogan and Cronan, 1997; Bao et al., 2002, 2003). None of the known natural sources of CPA are suitable for its commercial production. Therefore, it would be desirable to create an oilseed crop plant that accumulates high levels of CPA by heterologously expressing CPS in seeds. However, to date, heterologous expression of plant cyclopropane synthase genes has led to only approximately 1.0% CPA in the transgenic seeds (Yu et al., 2011).Two pathways for the biosynthesis of TAG exist in plants (Bates and Browse, 2012; Fig. 1). The de novo biosynthesis from glycerol-3-phosphate and acyl-CoA occurs via the Kennedy pathway and includes three acyltransferases: glycerol-2-phosphate acyltransferase, acyl-CoA:lysophosphatidic acid acyltransferase (LPAT), and acyl-CoA:diacylglycerol acyltransferase (DGAT; Kennedy, 1961). Alternatively, acyl-CoAs can be redirected from phosphatidylcholine (PC) via the action of a phospholipase C, choline phosphotransferase, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT; Hu et al., 2012; Lu et al., 2009), or phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). An acyl group can be released from PC to generate lysophosphatidylcholine (LPC) by the back reaction of acyl-CoA:LPC acyltransferase (Stymne and Stobart, 1984; Wang et al., 2012) or a phospholipase A/acyl-CoA synthase (Chen et al., 2011).Open in a separate windowFigure 1.Schematic representation of the plant TAG biosynthesis network. Acyl editing can provide PC-modified fatty acids for de novo diacylglycerol/TAG synthesis. ACS, acyl-CoA synthase; CPT, CDP-choline:diacylglycerol choline phosphotransferase; G3P, glycerol-3-phosphate; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPC acyltransferase, acyl-CoA:LPC acyltransferase; mFAS, modified fatty acid synthase (in this work, mFAS is CPS); PAP, phosphatidic acid phosphatase; PLA, phospholipase A; PLC, phospholipase C.LPAT is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid (LPA) into different phosphatidic acids (PAs) in diverse tissues. Membrane-associated LPAT activities, identified in bacteria, yeast, plant, and animal cells, catalyze the transfer of acyl groups from acyl-CoA to LPA to synthesize PA. In plants and other organisms, LPAT activities have been identified in the endoplasmic reticulum (Kim et al., 2005), plasma membrane (Bursten et al., 1991), and mitochondria (Zborowski and Wojtczak, 1969). In higher plants, endoplasmic reticulum-localized LPAT plays an essential role transferring fatty acid from CoA esters to the sn-2 position of LPA in the synthesis of PA, a key intermediate in the biosynthesis of membrane phospholipids and storage lipids in developing seeds (Maisonneuve et al., 2010). LPAT from developing seeds of flax (Linum usitatissimum), rape (Brassica napus), and castor bean preferentially incorporate oleoyl-CoA, weakly incorporate cyclopropane acyl-CoA, and were unable to incorporate methyl-branched acyl-CoA when presented with an equimolar mix of these potential substrates (Nlandu Mputu et al., 2009). Thus, LPAT activity from agronomic plants constitutes a potential bottleneck for the incorporation of branched chain acyl-CoA into PA. In this work, we investigate the utility of an LPAT from a cyclopropanoid-syntheizing plant, S. foetida, with respect to its ability to enhance CPA accumulation. In our efforts to enhance CPA accumulation in transgenic plants, we screened CPS genes from diverse sources and identified Escherichia coli CPS (EcCPS) as an effective enzyme for the production of CPA in plants. However, EcCPS is reported to prefer the sn-2 position of E. coli phospholipid (Hildebrand and Law, 1964), and the data presented here show that its expression primarily leads to the accumulation of CPA at the stereospecific numbering (sn)-1 position. Moreover, coexpression of S. foetida lysophosphatidic acid acyltransferase (SfLPAT) results in the incorporation of CPA at the sn-2 position of LPA. Thus, coexpression of EcCPS and SfLPAT enables a cycle that enriches the accumulation of CPA at both sn-1 and sn-2 positions of PC and increases the accumulation of CPA. This work underscores the utility of coexpressing an acyltransferase from mFA-accumulating species with mFA-synthesizing enzymes to help mitigate bottlenecks in mFA TAG synthesis.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.     

On the Inside

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.Plant cell expansion and anisotropic cell growth are driven by vacuolar turgor pressure and cell wall extensibility, which in a dynamic and restrictive manner direct cell morphogenesis (Baskin, 2005). Cellulose is the major load-bearing component of the cell wall and is thus a major determinant for anisotropic growth (Baskin, 2001). Cellulose is made up of β-1,4-linked glucan chains that may aggregate to form microfibrils holding 18 to 36 chains (Somerville, 2006; Fernandes et al., 2011; Jarvis, 2013; Newman et al., 2013; Thomas et al., 2013). In contrast to cell wall structural polysaccharides, including pectin and hemicellulose, which are synthesized by Golgi-localized enzymes, cellulose is synthesized at the plasma membrane (PM) by cellulose synthase complexes (CSCs; Somerville, 2006; Scheller and Ulvskov, 2010; Atmodjo et al., 2013). The cellulose synthases (CESAs) are the principal catalytic units of cellulose biosynthesis and in higher plants are organized into globular rosettes (Haigler and Brown, 1986). For their biosynthetic function, each primary cell wall CSC requires a minimum of three catalytic CESA proteins (Desprez et al., 2007; Persson et al., 2007).On the basis of observations that cellulose microfibrils align with cortical microtubules (MTs) and that MT disruption leads to a loss of cell expansion, it was hypothesized that cortical MTs guide the deposition and, therefore, the orientation of cellulose (Green, 1962; Ledbetter and Porter, 1963; Baskin, 2001; Bichet et al., 2001; Sugimoto et al., 2003; Baskin et al., 2004; Wasteneys and Fujita, 2006). Confocal microscopy of CESA fluorescent fusions has advanced our understanding of CESA trafficking and dynamics. CSCs are visualized as small particles moving within the plane of the PM, with an average velocity of approximately 200 to 400 nm min⁻¹. Their movement in linear tracks along cortical MTs (Paredez et al., 2006) supports the MT-cellulose alignment hypothesis.Our current understanding of cellulose synthesis suggests that CESAs are assembled into CSCs in either the endoplasmic reticulum (ER) or the Golgi apparatus and trafficked by vesicles to the PM (Bashline et al., 2014; McFarlane et al., 2014). The presence of CESAs in isolated Golgi and vesicles from the trans-Golgi network (TGN) has been established by proteomic studies (Dunkley et al., 2006; Drakakaki et al., 2012; Nikolovski et al., 2012; Parsons et al., 2012; Groen et al., 2014). Their localization at the TGN has been corroborated by electron microscopy and colocalization with TGN markers, such as vacuolar H⁺-ATP synthase subunit a1 (VHA-a1), and the Soluble NSF Attachment Protein Receptor (SNARE) protein SYNTAXIN OF PLANTS41 (SYP41), SYP42, and SYP61 (Crowell et al., 2009; Gutierrez et al., 2009; Drakakaki et al., 2012). A population of post-Golgi compartments carrying CSCs, referred to as microtubule-associated cellulose synthase compartments (MASCs) or small cellulose synthase compartments (SmaCCs), may be associated with MTs or actin filaments and are thought to be directly involved in either CSC delivery to, or internalization from, the PM (Crowell et al., 2009; Gutierrez et al., 2009).In addition to the CESAs, auxiliary proteins have been identified that play a vital role in the cellulose-synthesizing machinery. These include COBRA (Roudier et al., 2005), the endoglucanase KORRIGAN1 (KOR1; Lane et al., 2001; Lei et al., 2014b; Vain et al., 2014), and the recently identified POM-POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (POM2/CSI1; Gu et al., 2010; Bringmann et al., 2012). The latter protein functions as a linker between the cortical MTs and CSCs, as genetic lesions in POM2/CSI1 result in a lower incidence of coalignment between CSCs and cortical MTs (Bringmann et al., 2012). Given the highly regulated process of cellulose biosynthesis and deposition, it can be expected that many more accessory proteins participate in the delivery of CSCs and their interaction with MTs. Identification of these unique CSC-associated proteins can ultimately provide clues for the mechanisms behind cell growth and cell shape formation.Arabidopsis (Arabidopsis thaliana) mutants with defects in the cellulose biosynthetic machinery exhibit a loss of anisotropic growth, which results in organ swelling. This phenotype may be used as a diagnostic tool in genetic screens to identify cellulose biosynthetic and CSC auxiliary proteins (Mutwil et al., 2008). Chemical inhibitors complement genetic lesions to perturb, study, and control the cellular and physiological function of proteins (Drakakaki et al., 2009). A plethora of bioactive small molecules have been identified, and their analytical use contributes to our understanding of cellulose biosynthesis and CESA subcellular behavior (for review, see Brabham and Debolt, 2012). Small molecule treatment can induce distinct characteristic subcellular CESA patterns that can be broadly grouped into three categories (Brabham and Debolt, 2012). The first is characterized by the depletion of CESAs from the PM and their accumulation in cytosolic compartments, as observed for the herbicide isoxaben {N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethyoxybenzamide}, CGA 325615 [1-cyclohexyl-5-(2,3,4,5,6-pentafluorophe-noxyl)-1λ4,2,4,6-thiatriazin-3-amine], thaxtomin A (4-nitroindol-3-yl containing 2,5-dioxopiperazine), AE F150944 [N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-di-amine], and quinoxyphen [4-(2-bromo-4,5-dimethoxyphenyl)-3,4-dihydro-¹H-benzo-quinolin-2-one]; (Paredez et al., 2006; Bischoff et al., 2009; Crowell et al., 2009; Gutierrez et al., 2009; Harris et al., 2012). The second displays hyperaccumulation of CESAs at the PM, as seen for the herbicides dichlobenil (2,6-dichlorobenzonitrile) and indaziflam {N-[(1R,2S)-2,3-dihydro-2,6-dimethyl-¹H-inden-1-yl)-6-(1-fluoroethyl]-1,3,5-triazine-2,4-diamine} (Herth, 1987; DeBolt et al., 2007b; Brabham et al., 2014). The third exhibits disturbance of both CESAs and MTs and alters CESA trajectories at the PM, as exemplified by morlin (7-ethoxy-4-methylchromen-2-one; DeBolt et al., 2007a). Unique compounds inducing a phenotype combining CESA accumulation in intermediate compartments and disruption of CSC-MT interactions can contribute to both the identification of the accessory proteins linking CSCs with MTs and the vesicular delivery mechanisms of CESAs.In this study, we identified and characterized a unique cellulose deposition inhibitor, the small molecule CESA TRAFFICKING INHIBITOR (CESTRIN), which affects the localization pattern of CSCs and their interacting proteins in a unique way. The induction of cytoplasmic CESTRIN bodies might provide further clues for trafficking routes that carry CESAs to the PM.