Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis

The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan.     

Mutations in Multiple XXT Genes of Arabidopsis Reveal the Complexity of Xyloglucan Biosynthesis

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxt1 xxt5 and xxt2 xxt5) and a triple mutant (xxt1 xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxt1 xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls.     

Arabidopsis mannan synthase CSLA9 and glucan synthase CSLC4 have opposite orientations in the Golgi membrane

Several proteins encoded by the cellulose synthase-like (CSL) gene family are known to be processive glycan synthases involved in the synthesis of cell-wall polysaccharides. These include CSLA proteins, which synthesize β-(1→4)-linked mannans found in the walls of many plant species, and CSLC proteins, which are thought to synthesize the β-(1→4)-linked glucan backbone of xyloglucan, an abundant polysaccharide in the primary walls of many plants. CSLA and CSLC proteins are predicted to have multiple membrane spans, and their products (mannan and xyloglucan) accumulate in the Golgi lumen. Knowing where these proteins are located in the cell and how they are orientated in the membrane is important for understanding many aspects of mannan and xyloglucan biosynthesis. In this study, we investigate the subcellular localization and membrane protein topology of CSLA9 and CSLC4, the members of these two families that are most highly expressed in Arabidopsis. CSLA9 and CSLC4 are found predominantly in Golgi membranes, based on co-localization with the known ER/Golgi marker ERD2-YFP. The topology of epitope-tagged proteins was examined using protease protection experiments. Experiments were designed to determine the positions of both the protein termini and the active loop of the CSL proteins investigated. The topology of CSLA9 is characterized by an odd number of transmembrane domains (probably five) and an active site that faces the Golgi lumen. In contrast, CSLC4 has an even number of transmembrane domains (probably six) and an active site that faces the cytosol. The implications of these topologies on various aspects of hemicellulose biosynthesis are discussed.     

Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.     

IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-mediated plant transformation

Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin alpha proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin alpha family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin alpha members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin alpha members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed.     

Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs

NPR1 is required for systemic acquired resistance, and there are five NPR1 paralogs in Arabidopsis. Here we report knockout analysis of two of these, NPR3 and NPR4. npr3 single mutants have elevated basal PR-1 expression and the npr3 npr4 double mutant shows even higher expression. The double mutant plants also display enhanced resistance against virulent bacterial and oomycete pathogens. This enhanced disease resistance is partially dependent on NPR1, can be in part complemented by either wild-type NPR3 or NPR4, and is not associated with an elevated level of salicylic acid. NPR3 and NPR4 interact with TGA2, TGA3, TGA5 and TGA6 in yeast two-hybrid assays. Using bimolecular fluorescence complementation analysis, we show that NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts. Combined with our previous finding that basal PR-1 levels are also elevated in the tga2 tga5 tga6 triple mutant, we propose that NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance through their association with TGA2 and its paralogs.     

GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.     

ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis

The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germina- tion, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant- specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co- immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.     

Arabidopsis G-protein β subunit AGB1 interacts with NPH3 and is involved in phototropism

Heterotrimeric G proteins (Gα, Gβ and Gγ) have pleiotropic roles in plants, but molecular mechanisms underlying them remain to be elucidated. Here we show that Arabidopsis Gβ (AGB1) interacts with NPH3, a regulator of phototropism. Yeast two-hybrid assays, in vitro pull-down assays and bimolecular fluorescence complementation assays showed that AGB1 and NPH3 physically interact. NPH3-null mutation (nph3) is known to completely abolish hypocotyl phototropism. Loss-of-function mutants of AGB1 (agb1-1 and agb1-2) showed decreased hypocotyl phototropism, and agb1/nph3 double mutants showed no hypocotyl phototropism. These results suggest that AGB1 is involved in the NPH3-mediated regulation of phototropism.     

Interaction between methyl CpG-binding protein and ran GTPase during cell division in tobacco cultured cells

BACKGROUND AND AIMS: Methyl CpG-binding proteins are considered to play critical roles in epigenetic control of gene expression by recognizing and interacting with 5-methylcytosine (m(5)C) in eukaryotes. However, among 13 corresponding genes in Arabidopsis thaliana, designated as featuring a methyl-binding domain (MBD), only four have so far been shown actually to bind to m(5)C. One example, AtMBD5, was selected here to screen for interacting proteins. METHODS: Yeast two-hybrid assays were used for screening, and physical interaction was confirmed by pull-down and bimolecular fluorescence complementation (BiFC) assays. Cellular localization was analysed by fluorescence-tagged fusion proteins using tobacco (Nicotiana tabacum) cultured bright yellow 2 cells. KEY RESULTS: A gene finally identified was found to encode AtRAN3, a protein that belongs to the Ran GTPase family, which plays a critical role in nucleocytoplasmic transport and spindle bipolarization during cell division. AtMBD5 and AtRAN3 were clearly shown to interact in the nucleus by BiFC. On co-expression of AtMBD5-cyan fluorescence protein and yellow fluorescence protein-AtRAN3 in tobacco cells, both localized to the nucleus in the resting stage, migrating to the cytoplasm, primarily around chromatin, during mitosis, particularly at metaphase. CONCLUSIONS: These results suggest that AtMBD5 becomes localized to the vicinity of chromosomes with the aid of AtRAN3 during cell division, and may play an important role not only in maintenance of chromatin structures by binding to m(5)C, but also in progress through mitosis by detaching from m(5)C. The present findings also shed light on the physiological function of Ran GTPases, direct target proteins of which have not thus far been well defined, suggesting their key role in chromatin movements in plant cells.     

Interaction between serine carboxypeptidase-like protein TtGS5 and Annexin D1 in developing seeds of Triticum timopheevi

GS5 encoding a serine carboxypeptidase-like protein positively regulates grain size and weight through the regulation of grain width and filling and is helpful in improving cereal yields. Grain width variation determined by GS5 is associated with cell number and size, but the actual underlying mechanism is still unclear. Two orthologs of GS5, TtGS5-3A-G and TtGS5-3G-G, were cloned from the Triticum timopheevi accession no. CWI17006. To identify the proteins that interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains, we performed pull-down assays followed by liquid chromatography-mass spectrometry/mass spectrometry analysis. The analyses revealed 18 proteins were present in both the TtGS5-3A-G and TtGS5-3G-G interactomes. Among five candidates selected, only Annexin D1 interacted with both TtGS5-3A-G and TtGS5-3G-G in yeast. Annexin D1, TtGS5-3A-G, and TtGS5-3G-G were located on the cytoplasmic membranes of Arabidopsis protoplasts and onion epidermal cells, and interactions between Annexin D1 and TtGS5-3A-G, as well as TtGS5-3G-G, were shown by bimolecular fluorescence complementation assays. Annexin D1 was expressed widely in different tissues, and it co-expressed with TtGS5-3A-G/TtGS5-3G-G at the grain enlargement phase. These results indicated that Annexin D1 interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains. Together with the structural similarities of Annexin D1 to known fiber elongation factors, we proposed that TtGS5 might regulate the cell size by interacting with Annexin D1. The results provide significant new information for understanding the roles that GS5 plays in regulating grain size, which may be useful in improving crop yields.     

Inter-dependence of dimerization and organelle binding in myosin XI

Cytoplasmic streaming is a ubiquitous process in plant cells that is thought to be driven by the active movement of myosin XI motor proteins along actin filaments. These myosin motors bind to organelles through their C-terminal globular tail domain, although recent studies have also suggested a role for the central coiled-coil region during organelle binding. Here we have investigated the relationship between these two protein domains of MYA1, an Arabidopsis myosin XI, in a series of in vivo experiments demonstrating that dimerization of the coiled-coil region stabilizes organelle binding of the globular tail. Surprisingly, yeast two-hybrid assays, bimolecular fluorescence complementation, Förster resonance energy transfer and in vitro pull-down experiments all demonstrated that dimerization of the 174-residue MYA1 coiled coils by themselves was unstable. Furthermore, only the first of the two major coiled-coil segments in MYA1 contributed significantly to dimer formation. Interestingly, dimerization of myosin tail constructs that included the organelle-binding globular tail was stable, although the globular tails by themselves did not interact. This suggests an inter-dependent relationship between dimerization and organelle binding in myosin XI, whereby each process synergistically stimulates the other.     

Arabidopsis GT34 family contains five xyloglucan α-1,6-xylosyltransferases

The Arabidopsis genome includes seven family 34 glycosyltransferase (GT34) encoding genes. XXT1 and XXT2 have previously been shown to encode XyG α-1,6-xylosyltransferases, while knockout mutants of a third, XXT5, exhibit decreased XyG content, suggesting a similar activity. Here, we extend the study to the rest of the Arabidopsis GT34 genes in terms of biochemical activity and their roles in XyG biosynthesis. The enzyme activity of XXTs was investigated using recombinant protein expressed in E. coli. XyG analysis of single and double T-DNA insertion knockouts, together with overexpression of GT34s in selected mutant lines, provided detailed function of each gene. We reveal the activity of the third member of the GT34 gene family (XXT4) that exhibits xylosyltransferase activity. Double mutants for either xxt2 or xxt5 had a large impact on XyG content, structure and size distribution. Overexpression of the remaining member, XXT3, was able to restore XyG epitopes in xxt2, xxt5 and xxt2 xxt5 double knockouts, suggesting that it also encodes a protein with XXT activity. Our work demonstrates that five of the seven Arabidopsis GT34 genes encode XXT enzymes.     

Two Sec13p homologs, AtSec13A and AtSec13B, redundantly contribute to the formation of COPII transport vesicles in Arabidopsis thaliana

COPII vesicles mediate protein transport from ER to Golgi. Sec13 makes up lattice structure with Sec31 to form COPII vesicles. We analyzed expression of two Arabidopsis thaliana Sec13 homologs, AtSec13A and AtSec13B. AtSec13A was expressed in most parts of seedlings, while AtSec13B was partially expressed. Interaction of AtSec13A or AtSec13B with Sec31 homolog was demonstrated by bimolecular fluorescence complementation (BiFC).     

HSP70-3 Interacts with Phospholipase Dδ and Participates in Heat Stress Defense

Heat shock proteins (HSPs) function as molecular chaperones and are key components responsible for protein folding, assembly, translocation, and degradation under stress conditions. However, little is known about how HSPs stabilize proteins and membranes in response to different hormonal or environmental cues in plants. Here, we combined molecular, biochemical, and genetic approaches to elucidate the involvement of cytosolic HSP70-3 in plant stress responses and the interplay between HSP70-3 and plasma membrane (PM)-localized phospholipase Dδ (PLDδ) in Arabidopsis (Arabidopsis thaliana). Analysis using pull-down, coimmunoprecipitation, and bimolecular fluorescence complementation revealed that HSP70-3 specifically interacted with PLDδ. HSP70-3 bound to microtubules, such that it stabilized cortical microtubules upon heat stress. We also showed that heat shock induced recruitment of HSP70-3 to the PM, where HSP70-3 inhibited PLDδ activity to mediate microtubule reorganization, phospholipid metabolism, and plant thermotolerance, and this process depended on the HSP70-3–PLDδ interaction. Our results suggest a model whereby the interplay between HSP70-3 and PLDδ facilitates the re-establishment of cellular homeostasis during plant responses to external stresses and reveal a regulatory mechanism in regulating membrane lipid metabolism.

The heat shock protein 70-3 interacts with phospholipase Dδ to regulate microtubule organization, lipid metabolism, and plant thermotolerance in Arabidopsis.     

Protein lipoylation in mitochondria requires Fe–S cluster assembly factors NFU4 and NFU5

Plants have evolutionarily conserved NifU (NFU)-domain proteins that are targeted to plastids or mitochondria. “Plastid-type” NFU1, NFU2, and NFU3 in Arabidopsis (Arabidopsis thaliana) play a role in iron–sulfur (Fe–S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here, we confirmed that NFU4 and NFU5 are targeted to the mitochondria. The proteins were constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of NFU4 and NFU5 proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe–S cluster-containing respiratory complexes or aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth improved with elevated CO₂ treatment. In addition, pyruvate, 2-oxoglutarate, and branched-chain amino acids accumulated in nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, most likely providing Fe–S clusters to lipoyl synthase.

A pair of evolutionarily conserved proteins involved in iron–sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.     

A rice really interesting new gene H2‐type E3 ligase,OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programmes. Here, we characterized the rice (Oryza sativa) really interesting new gene (RING) H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na⁺ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared with wild type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na⁺ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.     

The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg2?-dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.