Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor

DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone alpha-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.     

Purification and characterization of a barley aleurone abscisic acid-binding protein

A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.     

Subcellular localization of nuclease in barley aleurone

The intracellular localization of an endonuclease (nuclease I) in barley aleurone responding to gibberellic acid was investigated by subcellular fractionation and immunocytochemistry with monoclonal and polyclonal antibodies. Organelle separations were performed with aleurone layers and protoplasts; immunefixations were carried out on protoplasts only. Nuclease was detected in fractions from isopycnic sucrose density gradients which were enriched in either endoplasmic reticulum or Golgi apparatus membranes. These two organelles were also labelled by the indirect immunogold method on thin sections. Intensive labelling of protein and developing vacuoles was observed. Therefore, as noted in other plants nuclease in barley is essentially a vacuolar enzyme.     

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

The structure and composition of aleurone grains in the barley aleurone layer

Summary Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.     

Loss-of-function of ACVR1 in osteoblasts increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1

BMPs (Bone morphogenetic proteins) such as BMP2 and BMP7 have been used about one decade as bone anabolic agents in orthopaedics. The BMP receptor ACVR1, which is a key receptor of BMP7, is expressed in bone. The pathological role of ACVR1 in humans has been reported: a point mutation in ACVR1 can cause fibrodysplasia ossificans progressiva (FOP) in which ectopic ossification occurs in skeletal muscles and deep connective tissues. The physiological function of ACVR1 in bone, however, is totally unknown. The purpose of this study is to investigate the endogenous role of ACVR1 in osteoblasts, one of the most dominant cell-types in bone. We generated Acvr1-null mice in an osteoblast-specific manner using an inducible Cre-loxP system. Surprisingly, we found that bone mass was increased in the Acvr1-null mice. Interestingly, canonical Wnt signaling was increased and expression levels of Wnt inhibitors Sost and Dkk1 were both suppressed in the null bones during the developmental stages. In addition, we confirmed that expression levels of both Sost and Dkk1 were upregulated by BMP7 dose-dependently in vitro. These results suggest that the Acvr1-deficiency can increase bone mass by activating Wnt signaling in which both Sost and Dkk1 expression levels are diminished. This study leads to a new concept of the BMP7-ACVR1-SOST/DKK1 axis in osteoblasts, in which BMP7 signaling through ACVR1 can reduce Wnt signaling via SOST/DKK1 and then inhibits osteogenesis. Although this concept is beyond the current known function of BMP7, it can explain the varied outcomes of BMP7 treatment. We believe BMP signaling can exhibit multifaceted effects by context and cell type.     

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Total protein release from barley aleurone layers as a bioassay for gibberellins

The effect of gibberellic acid on the secretion of proteins from barley (Hordeum vulgare L.) aleurone layers has been investigated for its suitability as a gibberellin bioassay. Concentrations from 10^–4 g/ml to 100 g/ml of GA₃ resulted in the release of proportionally increasing amounts of total protein. The release of proteins is not affected by indoleacetic acid and kinetin. This method has been applied and compared with the -amylase assay for the estimation of gibberellin in extracts of tomato fruits and maize seedlings.Abbreviations GA₃ gibberellic acid - IAA indoleactic acid - K kinetin     

The fine structure of barley aleurone cells

Summary The ultrastructural morphology of both dry and water-imbibed barley aleurone cells is described. The aleurone cell is characterized by the presence of numerous aleurone grains and spherosomes. In addition, it contains organelles typical of other plant cells including structures similar to microbodies, and rough endoplasmic reticulum characterized by the presence of numerous polyribosomes. It is inferred that the morphological specialization of aleurone cells is related to their biochemical specialization.Work supported by National Science Foundation grant GB5863. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.     

Hormonal and heat-stress regulation of protein synthesis in the aleurone layers of barley seeds

Barley aleurone cells have long served as a model system for studying the regulation of gene expression in plants. In this review we survey what is known about hormone-regulated gene expression in aleurone cells. We also describe the effects of heat stress on gene expression in this system, and speculate how the aleurone cell prioritizes its response between hormone-induced and environment-induced programs of gene expression.