MUCILAGE-MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis Seed Coat

Plant Physiology. Jan, 2004  |  Pubmed ID: 14701918

The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis. Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic rearrangement. We show that MUM4 encodes a putative NDP-l-rhamnose synthase, an enzyme required for the synthesis of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4 mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2 MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2, TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our results provide a framework for the regulation of mucilage production and secretory cell differentiation.

Plant Cuticular Lipid Export Requires an ABC Transporter

Science (New York, N.Y.). Oct, 2004  |  Pubmed ID: 15499022

A waxy protective cuticle coats all primary aerial plant tissues. Its synthesis requires extensive export of lipids from epidermal cells to the plant surface. Arabidopsis cer5 mutants had reduced stem cuticular wax loads and accumulated sheetlike inclusions in the cytoplasm of wax-secreting cells. These inclusions represented abnormal deposits of cuticular wax and resembled inclusions found in a human disorder caused by a defective peroxisomal adenosine triphosphate binding cassette (ABC) transporter. We found that the CER5 gene encodes an ABC transporter localized in the plasma membrane of epidermal cells and conclude that it is required for wax export to the cuticle.

Global Transcript Profiling of Primary Stems from Arabidopsis Thaliana Identifies Candidate Genes for Missing Links in Lignin Biosynthesis and Transcriptional Regulators of Fiber Differentiation

The Plant Journal : for Cell and Molecular Biology. Jun, 2005  |  Pubmed ID: 15918878

Different stages of vascular and interfascicular fiber differentiation can be identified along the axis of bolting stems in Arabidopsis. To gain insights into the metabolic, developmental, and regulatory events that control this pattern, we applied global transcript profiling employing an Arabidopsis full-genome longmer microarray. More than 5000 genes were differentially expressed, among which more than 3000 changed more than twofold, and were placed into eight expression clusters based on polynomial regression models. Within these, 182 upregulated transcription factors represent candidate regulators of fiber development. A subset of these candidates has been associated with fiber development and/or secondary wall formation and lignification in the literature, making them targets for functional studies and comparative genomic analyses with woody plants. Analysis of differentially expressed phenylpropanoid genes identified a set known to be involved in lignin biosynthesis. These were used to anchor co-expression analyses that allowed us to identify candidate genes encoding proteins involved in monolignol transport and monolignol dehydrogenation and polymerization. Similar analyses revealed candidate genes encoding enzymes that catalyze missing links in the shikimate pathway, namely arogenate dehydrogenase and prephenate aminotransferase.

Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem Epidermis

Plant Physiology. Dec, 2005  |  Pubmed ID: 16299169

All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 microg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 microg/cm2) to the lower (3 microg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of the lipid-related subset is presented.

A Role for Caleosin in Degradation of Oil-body Storage Lipid During Seed Germination

The Plant Journal : for Cell and Molecular Biology. Sep, 2006  |  Pubmed ID: 16961733

Caleosin is a Ca(2+)-binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing alpha- and delta-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid.

Synthesis of Enzymatically Active Human Alpha-L-iduronidase in Arabidopsis Cgl (complex Glycan-deficient) Seeds

Plant Biotechnology Journal. Mar, 2006  |  Pubmed ID: 17177794

As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T(2) seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 microg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 microg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans.

Influence of Poly(ethylene Glycol) Grafting Density and Polymer Length on Liposomes: Relating Plasma Circulation Lifetimes to Protein Binding

Biochimica Et Biophysica Acta. Jun, 2007  |  Pubmed ID: 17400180

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG(2000)) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG(2000). At this proportion of DSPE-PEG(2000), the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG(2000) in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.

The Distribution and Conformation of Very Long-chain Plant Wax Components in a Lipid Bilayer

The Journal of Physical Chemistry. B. Aug, 2007  |  Pubmed ID: 17608528

Plant wax contains long-chain alkanes and related components which are transported to the surface of the plant by specialized ABC transporter proteins. Here, we determine the distribution and conformation of three wax components, nonacosane, nonacosan-15-one, and nonacosan-15-ol, using unbiased and umbrella sampling molecular dynamics simulations. The molecules all partitioned to the center of the bilayer, with a free-energy difference of -70 kJ/mol between bulk water and the center of the bilayer for the alkane and -55 kJ/mol for the two more-polar molecules. All of the wax molecules were highly mobile in the bilayer, freely moving between opposite leaflets on a time scale of a few nanoseconds. Nonacosan-15-one and nonacosan-15-ol folded double to expose their hydrophilic group to the solvent, whereas nonacosane alternated between orientations spanning the full bilayer and orientations in the center of the bilayer.

Characterization of Arabidopsis ABCG11/WBC11, an ATP Binding Cassette (ABC) Transporter That is Required for Cuticular Lipid Secretion

The Plant Journal : for Cell and Molecular Biology. Nov, 2007  |  Pubmed ID: 17727615

ABCG11/WBC11, an ATP binding cassette (ABC) transporter from Arabidopsis thaliana, is a key component of the export pathway for cuticular lipids. Arabidopsis wbc11 T-DNA insertional knock-out mutants exhibited lipidic inclusions inside epidermal cells similar to the previously characterized wax transporter mutant cer5, with a similar strong reduction in the alkanes of surface waxes. Moreover, the wbc11 knock-out mutants also showed defects not present in cer5, including post-genital organ fusions, stunted growth and a reduction in cutin load on the plant surface. A mutant line previously isolated in a forward genetics screen, called permeable leaves 1 (pel1), was identified as an allele of ABCG11/WBC11. The double knock-out wbc11 cer5 exhibited the same morphological and biochemical phenotypes as the wbc11 knock-out. A YFP-WBC11 fusion protein rescued a T-DNA knock-out mutant and was localized to the plasma membrane. These results show that WBC11 functions in secretion of surface waxes, possibly by interacting with CER5. However, unlike ABCG12/CER5, ABCG11/WBC11 is important to the normal process of cutin formation.

The Cytochrome P450 Enzyme CYP96A15 is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

Plant Physiology. Nov, 2007  |  Pubmed ID: 17905869

Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.

Sealing Plant Surfaces: Cuticular Wax Formation by Epidermal Cells

Annual Review of Plant Biology. 2008  |  Pubmed ID: 18251711

The vital importance of plant surface wax in protecting tissue from environmental stresses is reflected in the huge commitment of epidermal cells to cuticle formation. During cuticle deposition, a massive flux of lipids occurs from the sites of lipid synthesis in the plastid and the endoplasmic reticulum to the plant surface. Recent genetic studies in Arabidopsis have improved our understanding of fatty acid elongation and of the subsequent modification of the elongated products into primary alcohols, wax esters, secondary alcohols, and ketones, shedding light on the enzymes involved in these pathways. In contrast, the biosynthesis of alkanes is still poorly understood, as are the mechanisms of wax transport from the site of biosynthesis to the cuticle. Currently, nothing is known about wax trafficking from the endoplasmic reticulum to the plasma membrane, or about translocation through the cell wall to the cuticle. However, a first breakthrough toward an understanding of wax export recently came with the discovery of ATP binding cassette (ABC) transporters that are involved in releasing wax from the plasma membrane into the apoplast. An overview of our present knowledge of wax biosynthesis and transport and the regulation of these processes during cuticle assembly is presented, including the evidence for coordination of cutin polyester and wax production.

Plant ABC Proteins--a Unified Nomenclature and Updated Inventory

Trends in Plant Science. Apr, 2008  |  Pubmed ID: 18299247

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

Cortical Microtubules Mark the Mucilage Secretion Domain of the Plasma Membrane in Arabidopsis Seed Coat Cells

Planta. May, 2008  |  Pubmed ID: 18309515

During their differentiation Arabidopsis thaliana seed coat cells undergo a brief but intense period of secretory activity that leads to dramatic morphological changes. Pectic mucilage is secreted to one domain of the plasma membrane and accumulates under the primary cell wall in a ring-shaped moat around an anticlinal cytoplasmic column. Using cryofixation/transmission electron microscopy and immunofluorescence, the cytoskeletal architecture of seed coat cells was explored, with emphasis on its organization, function and the large amount of pectin secretion at 7 days post-anthesis. The specific domain of the plasma membrane where mucilage secretion is targeted was lined by abundant cortical microtubules while the rest of the cortical cytoplasm contained few microtubules. Actin microfilaments, in contrast, were evenly distributed around the cell. Disruption of the microtubules in the temperature-sensitive mor1-1 mutant affected the eventual release of mucilage from mature seeds but did not appear to alter the targeted secretion of vesicles to the mucilage pocket, the shape of seed coat cells or their secondary cell wall deposition. The concentration of cortical microtubules at the site of high vesicle secretion in the seed coat may utilize the same mechanisms required for the formation of preprophase bands or the bands of microtubules associated with spiral secondary cell wall thickening during protoxylem development.

Analysis of the Golgi Apparatus in Arabidopsis Seed Coat Cells During Polarized Secretion of Pectin-rich Mucilage

The Plant Cell. Jun, 2008  |  Pubmed ID: 18523060

Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell.

Tracking Monolignols During Wood Development in Lodgepole Pine

Plant Physiology. Aug, 2008  |  Pubmed ID: 18550683

Secondary xylem (wood) formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and then reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm. This was done by feeding [(3)H]phenylalanine ([(3)H]Phe) to dissected cambium/developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, then rapidly freezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. Parallel experiments showed that radioactivity was incorporated into polymeric lignin and a methanol-soluble pool that was characterized by high-performance liquid chromatography. [(3)H]Phe was incorporated into expected lignin precursors, such as coniferyl alcohol and p-coumaryl alcohol, as well as pinoresinol. Coniferin, the glucoside of coniferyl alcohol, was detected by high-performance liquid chromatography but was not radioactively labeled. With light microscopy, radiolabeled phenylpropanoids were detected in the rays as well as the tracheids, with the two cell types showing differential sensitivity to inhibitors of protein translation and phenylpropanoid metabolism. Secondary cell walls of developing tracheids were heavily labeled when incubated with [(3)H]Phe. Inside the cell, cytoplasm was most strongly labeled followed by Golgi and low-vacuole label. Inhibitor studies suggest that the Golgi signal could be attributed to protein, rather than phenylpropanoid, origins. These data, produced with the best microscopy tools that are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols.

Identification of the Wax Ester Synthase/acyl-coenzyme A: Diacylglycerol Acyltransferase WSD1 Required for Stem Wax Ester Biosynthesis in Arabidopsis

Plant Physiology. Sep, 2008  |  Pubmed ID: 18621978

Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C(16) and C(18)) or very-long-chain (C(20) and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.

Perturbed Lignification Impacts Tree Growth in Hybrid Poplar--a Function of Sink Strength, Vascular Integrity, and Photosynthetic Assimilation

Plant Physiology. Nov, 2008  |  Pubmed ID: 18805953

The effects of reductions in cell wall lignin content, manifested by RNA interference suppression of coumaroyl 3'-hydroxylase, on plant growth, water transport, gas exchange, and photosynthesis were evaluated in hybrid poplar trees (Populus alba x grandidentata). The growth characteristics of the reduced lignin trees were significantly impaired, resulting in smaller stems and reduced root biomass when compared to wild-type trees, as well as altered leaf morphology and architecture. The severe inhibition of cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. In the reduced lignin trees, photosynthetic carbon assimilation and stomatal conductance were also greatly reduced, however, shoot xylem pressure potential and carbon isotope discrimination were higher and water-use efficiency was lower, inconsistent with water stress. Reductions in assimilation rate could not be ascribed to increased stomatal limitation. Starch and soluble sugars analysis of leaves revealed that photosynthate was accumulating to high levels, suggesting that the trees with substantially reduced cell wall lignin were not carbon limited and that reductions in sink strength were, instead, limiting photosynthesis.

A Unique Program for Cell Death in Xylem Fibers of Populus Stem

The Plant Journal : for Cell and Molecular Biology. Apr, 2009  |  Pubmed ID: 19175765

Maturation of the xylem elements involves extensive deposition of secondary cell-wall material and autolytic processes resulting in cell death. We describe here a unique type of cell-death program in xylem fibers of hybrid aspen (Populus tremula x P. tremuloides) stems, including gradual degradative processes in both the nucleus and cytoplasm concurrently with the phase of active cell-wall deposition. Nuclear DNA integrity, as determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and Comet (single-cell gel electrophoresis) assays, was compromised early during fiber maturation. In addition, degradation of the cytoplasmic contents, as detected by electron microscopy of samples fixed by high-pressure freezing/freeze substitution (HPF-FS), was gradual and resulted in complete loss of the cytoplasmic contents well before the loss of vacuolar integrity, which is considered to be the moment of death. This type of cell death differs significantly from that seen in xylem vessels. The loss of vacuolar integrity, which is thought to initiate cell degradative processes in the xylem vessels, is one of the last processes to occur before the final autolysis of the remaining cell contents in xylem fibers. High-resolution microarray analysis in the vascular tissues of Populus stem, combined with in silico analysis of publicly available data repositories, suggests the involvement of several previously uncharacterized transcription factors, ethylene, sphingolipids and light signaling as well as autophagy in the control of fiber cell death.

Arabidopsis LTPG is a Glycosylphosphatidylinositol-anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

The Plant Cell. Apr, 2009  |  Pubmed ID: 19366900

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.

Plant Cuticles Shine: Advances in Wax Biosynthesis and Export

Current Opinion in Plant Biology. Dec, 2009  |  Pubmed ID: 19864175

The plant cuticle is an extracellular lipid structure deposited over the aerial surfaces of land plants, which seals the shoot and protects it from biotic and abiotic stresses. It is composed of cutin polymer matrix and waxes, produced and secreted by epidermal cells. The use of forward and reverse genetic approaches in Arabidopsis has led to the identification of enzymes involved in fatty acid elongation and biosynthesis of wax components, as well as transporters required for lipid delivery to the cuticle. However, major questions concerning alkane formation, intracellular and extracellular wax transport, regulation of wax deposition, and assembly of cuticular components into a functional cuticle remain to be resolved.

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Journal of Integrative Plant Biology. Feb, 2010  |  Pubmed ID: 20377684

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides x P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofluorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.

ATP-binding Cassette Transporter G26 is Required for Male Fertility and Pollen Exine Formation in Arabidopsis

Plant Physiology. Oct, 2010  |  Pubmed ID: 20732973

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana). abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.

Arabidopsis ABCG Transporters, Which Are Required for Export of Diverse Cuticular Lipids, Dimerize in Different Combinations

The Plant Cell. Sep, 2010  |  Pubmed ID: 20870961

ATP binding cassette (ABC) transporters play diverse roles, including lipid transport, in all kingdoms. ABCG subfamily transporters that are encoded as half-transporters require dimerization to form a functional ABC transporter. Different dimer combinations that may transport diverse substrates have been predicted from mutant phenotypes. In Arabidopsis thaliana, mutant analyses have shown that ABCG11/WBC11 and ABCG12/CER5 are required for lipid export from the epidermis to the protective cuticle. The objective of this study was to determine whether ABCG11 and ABCG12 interact with themselves or each other using bimolecular fluorescence complementation (BiFC) and protein traffic assays in vivo. With BiFC, ABCG11/ABCG12 heterodimers and ABCG11 homodimers were detected, while ABCG12 homodimers were not. Fluorescently tagged ABCG11 or ABCG12 was localized in the stem epidermal cells of abcg11 abcg12 double mutants. ABCG11 could traffic to the plasma membrane in the absence of ABCG12, suggesting that ABCG11 is capable of forming flexible dimer partnerships. By contrast, ABCG12 was retained in the endoplasmic reticulum in the absence of ABCG11, indicating that ABCG12 is only capable of forming a dimer with ABCG11 in epidermal cells. Emerging themes in ABCG transporter biology are that some ABCG proteins are promiscuous, having multiple partnerships, while other ABCG transporters form obligate heterodimers for specialized functions.

SPIKE1 Signals Originate from and Assemble Specialized Domains of the Endoplasmic Reticulum

Current Biology : CB. Dec, 2010  |  Pubmed ID: 21109438

In the leaf epidermis, intricately lobed pavement cells use Rho of plants (ROP) small GTPases to integrate actin and microtubule organization with trafficking through the secretory pathway. Cell signaling occurs because guanine nucleotide exchange factors (GEFs) promote ROP activation and their interactions with effector proteins that direct the cell growth machineries. In Arabidopsis, SPIKE1 (SPK1) is the lone DOCK family GEF. SPK1 promotes polarized growth and cell-cell adhesion in the leaf epidermis; however, its mode of action in cells is not known. Vertebrate DOCK proteins are deployed at the plasma membrane. Likewise, current models place SPK1 activity and/or active ROP at the plant plasma membrane and invoke the localized patterning of the cortical cytoskeleton as the mechanism for shape control. In this paper, we find that SPK1 is a peripheral membrane protein that accumulates at, and promotes the formation of, a specialized domain of the endoplasmic reticulum (ER) termed the ER exit site (ERES). SPK1 signals are generated from a distributed network of ERES point sources and maintain the homeostasis of the early secretory pathway. The ERES is the location for cargo export from the ER. Our findings open up unexpected areas of plant G protein biology and redefine the ERES as a subcellular location for signal integration during morphogenesis.

Acyl-lipid Metabolism

The Arabidopsis Book / American Society of Plant Biologists. 2010  |  Pubmed ID: 22303259

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Self-immobilizing Fluorogenic Imaging Agents of Enzyme Activity

Angewandte Chemie (International Ed. in English). Jan, 2011  |  Pubmed ID: 21184404

Conserved Arabidopsis ECHIDNA Protein Mediates Trans-Golgi-network Trafficking and Cell Elongation

Proceedings of the National Academy of Sciences of the United States of America. May, 2011  |  Pubmed ID: 21512130

Multiple steps of plant growth and development rely on rapid cell elongation during which secretory and endocytic trafficking via the trans-Golgi network (TGN) plays a central role. Here, we identify the ECHIDNA (ECH) protein from Arabidopsis thaliana as a TGN-localized component crucial for TGN function. ECH partially complements loss of budding yeast TVP23 function and a Populus ECH complements the Arabidopsis ech mutant, suggesting functional conservation of the genes. Compared with wild-type, the Arabidopsis ech mutant exhibits severely perturbed cell elongation as well as defects in TGN structure and function, manifested by the reduced association between Golgi bodies and TGN as well as mislocalization of several TGN-localized proteins including vacuolar H(+)-ATPase subunit a1 (VHA-a1). Strikingly, ech is defective in secretory trafficking, whereas endocytosis appears unaffected in the mutant. Some aspects of the ech mutant phenotype can be phenocopied by treatment with a specific inhibitor of vacuolar H(+)-ATPases, concanamycin A, indicating that mislocalization of VHA-a1 may account for part of the defects in ech. Hence, ECH is an evolutionarily conserved component of the TGN with a central role in TGN structure and function.

The Endo-1,4-β-glucanase Korrigan Exhibits Functional Conservation Between Gymnosperms and Angiosperms and is Required for Proper Cell Wall Formation in Gymnosperms

The New Phytologist. Mar, 2012  |  Pubmed ID: 22150158

• The evolution of compositional polymers and their complex arrangement and deposition in the cell walls of terrestrial plants included the acquisition of key protein functions. • A membrane-bound endoglucanase, termed Korrigan (KOR), has been shown to be required for proper cellulose synthesis. To date, no extensive characterization of the gymnosperm KOR has been undertaken. • Characterization of the white spruce (Picea glauca) gene encoding KOR (PgKOR) shows conserved protein features such as polarized targeting signals and residues predicted to be essential for catalytic activity. The rescue of the Arabidopsis thaliana kor1-1 mutant by the expression of PgKOR suggests gene conservation, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development. Additionally, RNA interference of the endogenous gymnosperm gene substantially reduced growth and structural glucose content, but had no effect on cellulose ultrastructure. • Partial functional conservation of KOR in gymnosperms suggests that its role in cell wall synthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya, and shows that proteins contributing to proper cellulose deposition are important conserved features of vascular plants.

Plant Cell Wall Secretion and Lipid Traffic at Membrane Contact Sites of the Cell Cortex

Protoplasma. Feb, 2012  |  Pubmed ID: 22160188

Plant cell wall secretion is the result of dynamic vesicle fusion events at the plasma membrane. The importance of the lipid bilayer environment of the plasma membrane and its interactions with the endomembrane system through vesicle traffic are well recognized. Recent advances in yeast molecular biology and biochemistry lead us to re-examine the hypothesis that non-vesicular traffic of lipids through close contact sites of the plasma membrane and endoplasmic reticulum could also be important in plant cell wall biosynthesis. Non-vesicular traffic is the extraction and transfer of individual lipid molecules from a donor bilayer to a target bilayer, usually with the assistance of lipid transfer proteins.