The monolayer tapetum cells of the maturing f lowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by f lotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g͞cm 3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g͞cm 3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of f loral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

Cyclophilins belong to a large family of enzymes called “peptidyl prolyl isomerases” that assist protein folding and assembly. The cyclophilin CYP20–3 (also known as “ROC4”) is the only member of this group located in the stroma (soluble phase) of chloroplasts. In the present study we isolated mutant Arabidopsis plants defective in the CYP20–3 gene and found them to be hypersensitive to oxidative stress conditions created by high light levels, rose bengal, high salt levels, and osmotic shock. Chloroplast serine acetyltransferase (SAT1), a rate-limiting enzyme in cysteine biosynthesis, was identified as an interacting partner for CYP20–3 by protein interaction analyses. In the present experiments, SAT1 activity increased significantly under conditions of light and oxidative stress in concert with total thiols in wild-type plants. By contrast, these parameters changed only marginally in experiments with the cyp20–3 mutant, suggesting that CYP20–3 links light and stress to SAT1 activity and cysteine biosynthesis. In further support of this conclusion, our analyses showed that the salt-hypersensitive phenotype of the mutant developed under illumination and not in the dark. Together with the earlier report that CYP20–3 foldase activity is enhanced by thioredoxin-mediated reduction, our findings suggest that CYP20–3 links photosynthetic electron transport and redox regulation to the folding of SAT1, thereby enabling the cysteine-based thiol biosynthesis pathway to adjust to light and stress conditions.

Although it is well known that Tyr phosphatases play a critical role in signal transduction in animal cells, little is understood of the functional significance of Tyr phosphatases in higher plants. Here, we describe the functional analysis of an Arabidopsis gene ( AtPTEN1 ) that encodes a Tyr phosphatase closely related to PTEN, a tumor suppressor in animals. The recombinant AtPTEN1 protein, like its homologs in animals, is an active phosphatase that dephosphorylates phosphotyrosine and phosphatidylinositol substrates. RNA gel blot analysis and examination of promoter-reporter constructs in transgenic Arabidopsis plants revealed that the AtPTEN1 gene is expressed exclusively in pollen grains during the late stage of development. Suppression of AtPTEN1 gene expression by RNA interference caused pollen cell death after mitosis. We conclude that AtPTEN1 is a pollen-specific phosphatase and is essential for pollen development.

Magnesium is an abundant divalent cation in plant cells and plays a critical role in many physiological processes. We have previously described the identification of a 10-member Arabidopsis gene family encoding putative magnesium transport (MGT) proteins. Here, we report that a member of the MGT family, AtMGT5, functions as a dual-functional Mg-transporter that operates in a concentration-dependent manner, namely it serves as a Mg-importer at micromolar levels and facilitates the efflux in the millimolar range. The AtMGT5 protein is localized in the mitochondria, suggesting that AtMGT5 mediates Mg-trafficking between the cytosol and mitochondria. The AtMGT5 gene was exclusively expressed in anthers at early stages of flower development. Examination of two independent T-DNA insertional mutants of AtMGT5 gene demonstrated that AtMGT5 played an essential role for pollen development and male fertility. This study suggests a critical role for Mg(2+) transport between cytosol and mitochondria in male gametogenesis in plants.

SummaryIn Brassica anthers during microsporogenesis, the tapetum cells contain two abundant lipid-rich organelles, the tapetosomes possessing oleosins and triacylglycerols (TAGs), and the elaioplasts having unique polypeptides and neutral esters. B. campestris, for its simplicity of possessing only the AA genome and one predominant oleosin of 45 kDa, was studied. In the developing anthers, the lipids and proteins of the tapetosomes and elaioplasts were concomitantly accumulated but selectively degraded or retained. Upon incubation of isolated tapetosomes in a pH-5 medium, the predominant 45 kDa oleosin underwent selective enzymatic proteolysis to a 37 kDa fragment, which was not further hydrolyzed upon prolonged incubation. The unreacted 45 kDa oleosin was retained in the organelles, whereas the 37 kDa fragment was released to the exterior. The fragment would become the predominant 37 kDa polypeptide in the pollen coat. Isolated tapetosomes did not undergo hydrolysis of the TAGs upon incubation in media of diverse pHs. An alkaline lipase in the soluble fraction of the anther extract was presumed to be the enzyme that would hydrolyze the tapetosome TAGs, which disappeared in the anthers during development. The tapetum elaioplasts contained several unique polypeptides of 31-36 kDa. The gene encoding a 32 kDa polypeptide was cloned, and its deduced amino acid sequence was homologous to those of two proteins known to be present on the surface of fibrils in chromoplasts. Upon incubation of isolated elaioplasts in media of diverse pHs, the organelle polypeptides were degraded completely and most rapidly at pH 5, whereas the neutral esters remained unchanged; these neutral esters would become the major lipid components of the pollen coat. The findings show that the constituents of the two major tapetum organelles underwent very different paths of degradation, or modification, and transfer to the pollen surface.

In seeds, the subcellular storage oil bodies have a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We used two maize (Zea mays L.) strains having diverse kernel (seed) oil contents to study the effects of varying the oil and oleosin contents on the structure of the oil bodies. Illinois High Oils (IHO, 15% w/w oils) and Illinois Low Oils (ILO, 0.5%) maize kernels were the products of breeding for diverse oil contents for about 100 generations. In both maize strains, although the genes for oil synthesis had apparently been modified drastically, the genes encoding oleosins appeared to be unaltered, as revealed by Southern blot analyses of the three oleosin genes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting of the oleosins. In addition, both strains contained the same three oleosin isoforms of a defined proportion, and both accumulated oils and oleosins coordinately. Oleosins in both strains were restricted to the oil bodies, as shown by analyses of the various subcellular fractions separated by sucrose-density-gradient centrifugation. Electron microscopy of the embryos and the isolated organelles revealed that the oil bodies in IHO were larger and had a spherical shape, whereas those in ILO were smaller and had irregular shapes. We conclude that in seeds, oleosin genes are expressed independent of the oil contents, and the size and shape of the oil bodies are dictated by the ratio of oils to oleosins synthesized during seed maturation. The extensive breeding for diverse oil contents has not altered the apparent mechanism of oil-body synthesis and the occurrence of hetero-dimer or -multimer of oleosin isoforms on the oil bodies.

SummaryReversible protein phosphorylation is the most common mechanism for cellular regulation in eukaryotic systems. Indeed, approximately 5% of the Arabidopsis genome encodes protein kinases and phosphatases. Among the thousands of such enzymes, only a small fraction has been examined experimentally. Studies have demonstrated that Ser/Thr phosphorylation and dephosphorylation play a key role in the regulation of plant physiology and development. However, function of tyrosine phosphorylation, despite the overwhelming importance in animals, has not been systematically studied in higher plants. As a result, it is still controversial whether tyrosine phosphorylation is important in plant signal transduction. Recently, the first two protein tyrosine phosphatases (PTPs) from a higher plant were characterized. A diverse group of genes encoding putative PTPs have been identified from the Arabidopsis genome sequence databases. Genetic analyses of various PTPs are underway and preliminary results have provided evidence that these PTPs serve critical functions in plant responses to stress signals and in plant development.© New Phytologist (2001) 151 : 155 -164

Yeast (Saccharomyces cerevisiae) has been used extensively as a heterologous eukaryotic system to study the intracellular targeting of proteins to different organelles. The lipid bodies in yeast have not been previously subjected to such studies. These organelles are functionally equivalent to the subcellular storage oil bodies in plant seeds. A plant oil body has a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We tested whether plant oleosin could be correctly targeted to the lipid bodies in transformed yeast. The coding region of a maize (Zea mays L.) oleosin gene was incorporated into yeast high copy and low copy number plasmids in which its expression was under the control of GAL1 promoter. Yeast strains transformed with these plasmids produced oleosin when grown in a medium containing galactose but not glucose. The oleosin produced in yeast had a molecular mass slightly higher than that of the native protein in maize. Oleosin accumulated concomitantly with the storage lipids during growth of the transformed yeast, and it was not secreted. Subcellular fractionation of the cell extracts obtained by two different cell breakage procedures revealed that the oleosin was largely restricted to the lipid bodies. Oleosin apparently did not affect the lipid contents and composition of the transformed yeast lipid bodies but replaced some of the native proteins associated with the organelles. Immunocytochemistry of the transformed yeast cells showed that the oleosin was present mostly on the periphery of the lipid bodies. Oleosin isolated from maize or transformed yeast strain, alone or in the presence of phospholipids or SDS, did not bind to the yeast lipid bodies in vitro. We conclude that plant oleosin is correctly targeted to the lipid bodies in transformed yeast and that yeast may be used as a heterologous system to dissect the intracellular targeting signals in the oleosin.Diverse organisms store lipids in subcellular particles as food reserves that will be mobilized during a forthcoming period of active metabolism. These lipid particles can be found in seeds, pollens, spores, and vegetative organs of plants (1). They are also present in the brown adipose (2) and other tissues of mammals (3), the eggs of some nematodes (4), and unicellular organisms such as yeast (5, 6), Euglena (7), and algae (8). Of all these subcellular storage lipid particles, those from plant seeds have been studied most extensively.The seeds of many plant species store triacylglycerols (TAGs) 1 as food reserves for germination and growth of the seedlings (1, 3). The TAGs constitute about 5-40% of the total seed dry weight. They are present in small discrete subcellular organelles called oil bodies (lipid bodies, oleosomes, and spherosomes). The spherical organelles have diameters of about 0.6 -2.0 m, depending on the plant species in which they occur. Each oil body contains a TAG matrix surrounded by a layer of phospholipids (PL) embedded with proteins ter...