Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as alpha-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Differential expression of two functional serine/threonine protein kinases from soybean that have an unusual acidic domain at the carboxy terminus

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::beta-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/ antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses.     

A novel blue light- and abscisic acid-inducible gene of Arabidopsis thaliana encoding an intrinsic membrane protein

Continuous irradiation with blue light (400-500 nm) induces flower formation in plantlets of Arabidopsis thaliana (C24) while red light (600-700 nm) is ineffective. This observation started a search for genes that are activated by blue light and initiate the morphogenic programme leading to flower formation. Several genes were identified via their cDNAs. From these clone AthH2, with an open reading frame for a hydrophobic 30.5 kDa polypeptide, was selected for further characterization of the corresponding gene. From a genomic library a DNA fragment of about 6.4 kb was isolated, comprising the coding region as well as 5'-upstream and 3'-downstream flanking segments. The coding region is composed of four exons, which specify a polypeptide of 286 amino acids. Several potential regulatory elements were found between position -670 and -1140 including GA and ABA sequence motifs. The latter could account for the observed induction of the AthH2 gene by ABA. Southern blot analysis of Arabidopsis genomic DNA suggests that the AthH2 gene is encoded by a single-copy gene. Hydropathy plots and secondary structure analysis of the putative polypeptide predict six membrane-spanning domains implicating a function as transmembrane channel protein. It displays significant homology with the proteins TR7a of pea (82%) and RD 28 of A. thaliana (68%).     

Signalling of abscisic acid to regulate plant growth

Abscisic acid (ABA) mediated growth control is a fundamental response of plants to adverse environmental cues. The linkage between ABA perception and growth control is currently being unravelled by using different experimental approaches such as mutant analysis and microinjection experiments. So far, two protein phosphatases, ABI1 and ABI2, cADPR, pH, and Ca2+ have been identified as main components of the ABA signalling pathway. Here, the ABA signal transduction pathway is compared to signalling cascades from yeast and mammalian cells. A model for a bifurcated ABA signal transduction pathway exerting a positive and negative control mechanism is proposed.     

C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes

A carrot gene homologous to the ABI3 gene of Arabidopsis was isolated from a carrot somatic embryo cDNA library and designated C-ABI3. The sequence of C-ABI3 was very similar to those of ABI3 of Arabidopsis and VP1 of maize in certain conserved regions. The expression of C-ABI3 was detected specifically in embryogenic cells, somatic embryos and developing seeds. Thus, expression of C-ABI3 was limited to tissues that acquired desiccation tolerance in response to endogenous or exogenous abscisic acid (ABA). Endogenous levels of ABA in seeds increased transiently and then desiccation of seeds started. The expression of C-ABI3 in developing seeds was observed prior to the increase in levels of endogenous ABA that was followed by desiccation of seeds. In transgenic mature leaves in which C-ABI3 was ectopically expressed, expression of ECP31, ECP63 and ECP40 was induced by treatment with ABA, which indicates that the expression of ECP genes was controlled by the pathway(s) that involved C-ABI3 and ABA. This suggests that C-ABI3 has the same function as VP1/ABI3 factor in carrot somatic embryos.     

Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst

In order to understand the molecular mechanisms which are responsible for desiccation tolerance in the resurrection plant Craterostigma plantagineum Hochst. a thorough analysis of the CDeT11-24 gene family was performed. CDeT11-24 comprises a small gene family whose genes are expressed in response to dehydration, salt stress and abscisic acid (ABA) treatment in leaves. The gene products are constitutively expressed in roots and disappear only when the plants are transferred to water. It is therefore suggested that the proteins are involved in sensing water status. The predicted proteins are very hydrophilic; they share some features with late-embryogenesis-abundant proteins, and sequence similarities were found with two ABA- and drought-regulated Arabidopsis genes. The analysis of beta-glucuronidase reporter genes driven by the CDeT11-24 promoter showed high activity in mature seeds in both transgenic Arabidopsis and tobacco. In vegetative tissues the promoter activity in response to ABA was restricted to young Arabidosis seedlings. The responsiveness to ABA during later developmental stages was regained in the presence of the Arabidopsis gene product ABI3. Dehydration-induced promoter activity was only observed in Arabidopsis leaves at a particular developmental stage. This analysis indicates that some components in the signal transduction pathway of the resurrection plant are not active in tobacco or Arabidopsis.     

High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells

Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 x 10(7) CFU/microgram of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10(6) CFU/microgram of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.     

Effects of age on activity patterns after coronary artery bypass surgery

By screening for new seed color mutations, we have identified a new gene, pale aleurone color1 (pac1), which when mutated causes a reduction in anthocyanin pigmentation. The pac1 gene is not allelic to any known anthocyanin biosynthetic or regulatory gene. The pac1-ref allele is recessive, nonlethal, and only reduces pigment in kernels, not in vegetative tissues. Genetic and molecular evidence shows that the pac1-ref allele reduces pigmentation by reducing RNA levels of the biosynthetic genes in the pathway. The mutant does not reduce the RNA levels of either of the two regulatory genes, b and c1. Introduction of an anthocyanin structural gene promoter (a1) driving a reporter gene into maize aleurones shows that pac1-ref kernels have reduced expression resulting from the action of the a1 promoter. Introduction of the reporter gene with constructs that express the regulatory genes b and c1 or the phlobaphene pathway regulator p shows that this reduction in a1-driven expression occurs in both the presence and absence of these regulators. Our results imply that pac1 is required for either b/c1 or p activation of anthocyanin biosynthetic gene expression and that pac1 acts independently of these regulatory genes.     

The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes

In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.     

Inhibition of the iron-induced ZmFer1 maize ferritin gene expression by antioxidants and serine/threonine phosphatase inhibitors

Two pathways have been implicated in the regulation of maize ferritin synthesis in response to iron. One of them involves the plant hormone abscisic acid (ABA) and controls the expression of ZmFer2 gene(s). Another pathway, ABA-independent, has been characterized in a de-rooted maize plantlet system and involves an oxidative step. The ZmFer1 maize ferritin gene is not regulated by ABA, and it is shown in this paper that the corresponding mRNA accumulates in de-rooted maize plantlets and BMS (Black Mexican Sweet) maize cell suspension cultures in response to iron via the oxidative pathway described previously. To investigate ZmFer1 gene regulation further, the BMS cell system has been used to develop a transient expression assay using a ZmFer1-beta-glucuronidase fusion. Both iron induction and antioxidant inhibition of ZmFer1 gene expression were observed in this system. Using Northern blot analysis and transient expression experiments, it was shown that both okadaic acid and calyculin A, two serine/ threonine phosphatase inhibitors, specifically inhibit ZmFer1 gene expression. These data indicate that an okadaic acid-sensitive protein phosphatase activity is involved in the regulation of the ZmFer1 ferritin gene in maize cells, and this activity is required for iron-induced expression of this gene.     

Acute cocaine effects on stereotype and defense: an ethoexperimental approach

Germinated barley foodstuff (GBF), derived from the aleurone and scutellum fractions of germinated barley, is rich in glutamine and low-lignified hemicellulose, and increases mucosal protein, RNA, and DNA content in the intestine when fed to normal rats. The aim of this study was to evaluate the effects of feeding GBF or germinated gramineous seeds on experimental ulcerative colitis. Sprague-Dawley rats that received 3% dextran sulfate sodium in their diets were used as an experimental colitis model. The effects of sulfasalazine, a drug used to treat inflammatory bowel disease, were compared with those of GBF. After rats had consumed diets containing GBF or various aleurone and scutellum fractions, mucosal damage; the content of mucosal protein, RNA, and DNA in the colo-rectum; and serum interleukin-8 and alpha1-acid glycoprotein levels were assessed. GBF and germinated seeds more effectively prevented bloody diarrhea and mucosal damage in colitis compared with controls and rats receiving sulfasalazine, but non-germinated samples did not have a protective effect. GBF increased mucosal protein and RNA content in the colitis model. The consumption of GBF appears to prevent inflammation in a colitis model, and its effect seems to be related to the germination process. GBF and germinated seeds have the potential to serve as nutritional therapy for ulcerative colitis.     

Synthesis and radiopharmacology of O-(2-[18F]fluoroethyl)-L-tyrosine for tumor imaging

The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22 degrees C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways.