Signaling from an altered cell wall to the nucleus mediates sugar-responsive growth and development in Arabidopsis thaliana

Sugars such as glucose function as signal molecules that regulate gene expression, growth, and development in plants, animals, and yeast. To understand the molecular mechanisms of sugar responses, we isolated and characterized an Arabidopsis thaliana mutant, high sugar response8 (hsr8), which enhances sugar-responsive growth and gene expression. Light-grown hsr8 plants exhibited increased starch and anthocyanin and reduced chlorophyll content in response to glucose treatment. Dark-grown hsr8 seedlings showed glucose-hypersensitive hypocotyl elongation and development. The HSR8 gene, isolated using map-based cloning, was allelic to the MURUS4 (MUR4) gene involved in arabinose synthesis. Dark-grown mur1 and mur3 seedlings also exhibited similar sugar responses to hsr8/mur4. The sugar-hypersensitive phenotypes of hsr8/mur4, mur1, and mur3 were rescued by boric acid, suggesting that alterations in the cell wall cause hypersensitive sugar-responsive phenotypes. Genetic analysis showed that sugar-hypersensitive responses in hsr8 mutants were suppressed by pleiotropic regulatory locus1 (prl1), indicating that nucleus-localized PRL1 is required for enhanced sugar responses in hsr8 mutant plants. Microarray analysis revealed that the expression of many cell wall-related and sugar-responsive genes was altered in mur4-1, and the expression of a significant proportion of these genes was restored to wild-type levels in the mur4-1 prl1 double mutant. These findings reveal a pathway that signals changes in the cell wall through PRL1 to altered gene expression and sugar-responsive metabolic, growth, and developmental changes.     

Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

Characterization of active oxygen-producing proteins in response to hypo-osmolarity in tobacco and Arabidopsis cell suspensions: identification of a cell wall peroxidase

The oxidative response induced by hypo-osmolarity is characterized in tobacco and Arabidopsis cells in order to identify the corresponding active oxygen-producing proteins. The pharmacological profiles of the oxidative responses were clearly different in the two plant materials, leading to the identification of distinct active oxygen producers in tobacco and Arabidopsis cells. In tobacco cells, a 100 kDa protein, localized in the plasma membrane, was demonstrated to produce active oxygen in the presence of NADPH. This production can be activated by fatty acids and is strongly depressed by diphenylene iodonium, as measured by an in vivo response. In Arabidopsis, 30 kDa and 34 kDa proteins localized in the cell wall were shown to be able to produce active oxygen in the presence of cofactors and the production is prevented by peroxidase inhibitors, as is the in vivo response. The two purified proteins were identified by mass spectrometry and both correspond to the peroxidase gene At5g64120.     

A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE–tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.     

Interspecies compatibility of the anther specific cell wall invertase promoters from Arabidopsis and tobacco for generating male sterile plants

Histochemical GUS-staining and fluorometric analyses revealed strong tissue specific activities of the cell wall invertase promoters Nin88 from Nicotiana tabacum and AtcwINV2 from Arabidopsis thaliana that are restricted tightly to anthers and pollen, respectively. Both in A. thaliana and N. tabacum repression of invertase activity by anther specific RNA-interference turned out to be an efficient method to circumvent carbohydrate supply of the symplastically isolated pollen with subsequent strong decrease of pollen germination ability and seed setting. In the case of tobacco, comparable results were also obtained by expressing a proteinaceous invertase inhibitor, whereas this approach was less efficient in Arabidopis. The present study revealed that anther specific interference with invertase-activity in order to generate male sterile plants can be applied to members of the two different plant families Solanaceae (N. tabacum) and Brassicaceae (A. thalaina) and the strategy seems to be a general tool for practical application in hybrid breeding or as biological safety precautions. To elucidate the compatibility of the isolated promoters beyond plant families, we transferred the regulatory sequences into the respectively heterologous systems, i.e. the Nin88 promoter into Arabidopsis and the AtcwINV2 promoter into tobacco. The specificities of both promoters are maintained in the heterologous backgrounds, but their activities are strongly reduced as GUS-stainings of flowers and pollen revealed and fluorometrical quantification confirmed.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.     

Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco

The secretory pathway in plants involves sustained traffic to the cell wall, as matrix components, polysaccharides and proteins reach the cell wall through the endomembrane system. We studied the secretion pattern of cell-wall proteins in tobacco protoplasts and leaf epidermal cells using fluorescent forms of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2). The two most representative protein fusions, secGFP-PMEI1 and PGIP2-GFP, reached the cell wall by passing through ER and Golgi stacks but using distinct mechanisms. secGFP-PMEI1 was linked to a glycosylphosphatidylinositol (GPI) anchor and stably accumulated in the cell wall, regulating the activity of the endogenous pectin methylesterases (PMEs) that are constitutively present in this compartment. A mannosamine-induced non-GPI-anchored form of PMEI1 as well as a form (PMEI1-GFP) that was unable to bind membranes failed to reach the cell wall, and accumulated in the Golgi stacks. In contrast, PGIP2-GFP moved as a soluble cargo protein along the secretory pathway, but was not stably retained in the cell wall, due to internalization to an endosomal compartment and eventually the vacuole. Stable localization of PGIP2 in the wall was observed only in the presence of a specific fungal endopolygalacturonase ligand in the cell wall. Both secGFP-PMEI1 and PGIP2-GFP sorting were distinguishable from that of a secreted GFP, suggesting that rigorous and more complex controls than the simple mechanism of bulk flow are the basis of cell-wall growth and differentiation.     

A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

CCR1, an enzyme required for lignin biosynthesis in Arabidopsis,mediates cell proliferation exit for leaf development

After initiation, leaves first undergo rapid cell proliferation. During subsequent development, leaf cells gradually exit the proliferation phase and enter the expansion stage, following a basipetally ordered pattern starting at the leaf tip. The molecular mechanism directing this pattern of leaf development is as yet poorly understood. By genetic screening and characterization of Arabidopsis mutants defective in exit from cell proliferation, we show that the product of the CINNAMOYL CoA REDUCTASE (CCR1) gene, which is required for lignin biosynthesis, participates in the process of cell proliferation exit in leaves. CCR1 is expressed basipetally in the leaf, and ccr1 mutants exhibited multiple abnormalities, including increased cell proliferation. The ccr1 phenotypes are not due to the reduced lignin content, but instead are due to the dramatically increased level of ferulic acid (FeA), an intermediate in lignin biosynthesis. FeA is known to have antioxidant activity, and the levels of reactive oxygen species (ROS) in ccr1 were markedly reduced. We also characterized another double mutant in CAFFEIC ACID O‐METHYLTRANSFERASE (comt) and CAFFEOYL CoA 3‐O‐METHYLTRANSFERASE (ccoaomt), in which the FeA level was dramatically reduced. Cell proliferation in comt ccoaomt leaves was decreased, accompanied by elevated ROS levels, and the mutant phenotypes were partially rescued by treatment with FeA or another antioxidant (N‐acetyl‐l ‐cysteine). Taken together, our results suggest that CCR1, FeA and ROS coordinate cell proliferation exit in normal leaf development.     

A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis

A crucial role for sterols in plant growth and development is underscored by the identification of three Arabidopsis sterol biosynthesis mutants that exhibit embryonic defects: fackel (fk), hydra1 (hyd1), and sterol methyltransferase 1/cephalopod (smt1/cph). We have taken a dual approach of sterol profiling and ultrastructural analysis to investigate the primary defects underlying the mutant phenotypes. Comprehensive gas chromatography GC-MS analysis of hyd1 in comparison to fk reveals an abnormal accumulation of unique sterol intermediates in each case. Sterol profiling of the fk hyd1 double mutant provides genetic evidence that FK C-14 reductase acts upstream of HYD1 C-8,7 isomerase. Despite distinct differences in sterol profiles, fk and hyd1 as well as smt1/cph share ultrastructural features such as incomplete cell walls and aberrant cell wall thickenings in embryonic and post-embryonic tissues. The common defects are coupled with ectopic callose and lignin deposits. We show that all three mutants exhibit a deficiency in cellulose, but are not reduced in pectin and sugars of the cell wall and cytosol. The sterol biosynthesis inhibitors 15-azasterol and fenpropimorph also cause cell wall gaps in dividing root cells and a reduction in bulk cellulose, corroborating that the cell wall abnormalities are due to altered sterol composition. Our results demonstrate that sterols are crucial for cellulose synthesis in the building of the plant cell wall.     

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.     

Mutation of SAC1, an Arabidopsis SAC domain phosphoinositide phosphatase, causes alterations in cell morphogenesis, cell wall synthesis, and actin organization

SAC (for suppressor of actin) domain proteins in yeast and animals have been shown to modulate the levels of phosphoinositides, thereby regulating several cellular activities such as signal transduction, actin cytoskeleton organization, and vesicle trafficking. Nine genes encoding SAC domain-containing proteins are present in the Arabidopsis thaliana genome, but their roles in plant cellular functions and plant growth and development have not been characterized. In this report, we demonstrate the essential roles of one of the Arabidopsis SAC domain proteins, AtSAC1, in plant cellular functions. Mutation of the AtSAC1 gene in the fragile fiber7 (fra7) mutant caused a dramatic decrease in the wall thickness of fiber cells and vessel elements, thus resulting in a weak stem phenotype. The fra7 mutation also led to reduced length and aberrant shapes in fiber cells, pith cells, and trichomes and to an alteration in overall plant architecture. The AtSAC1 gene was found to be expressed in all tissues in elongating organs; however, it showed predominant expression in vascular tissues and fibers in nonelongating parts of stems. In vitro activity assay demonstrated that AtSAC1 exhibited phosphatase activity toward phosphatidylinositol 3,5-biphosphate. Subcellular localization studies showed that AtSAC1 was colocalized with a Golgi marker. Truncation of the C terminus by the fra7 mutation resulted in its localization in the cytoplasm but had no effect on phosphatase activity. Furthermore, examination of the cytoskeleton organization revealed that the fra7 mutation caused the formation of aberrant actin cables in elongating cells but had no effect on the organization of cortical microtubules. Together, these results provide genetic evidence that AtSAC1, a SAC domain phosphoinositide phosphatase, is required for normal cell morphogenesis, cell wall synthesis, and actin organization.     

A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions.

Regulation of the cell death program involves physical interactions between different members of the Bcl-2 family that either promote or suppress apoptosis. The Bcl-2 homolog, Bak, promotes apoptosis and binds anti-apoptotic family members including Bcl-2 and Bcl-xL. We have identified a domain in Bak that is both necessary and sufficient for cytotoxic activity and binding to Bcl-xL. Sequences similar to this domain were identified in Bax and Bip1, two other proteins that promote apoptosis and interact with Bcl-xL, and were likewise critical for their capacity to kill cells and bind Bcl-xL. Thus, the domain is of central importance in mediating the function of multiple cell death-regulatory proteins that interact with Bcl-2 family members.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.