Plant metabolism: enzyme regulation by 14-3-3 proteins.

14-3-3 proteins have been found to regulate the plant enzyme nitrate reductase by reversible phosphoserine binding. Plant plasma-membrane H(+)-ATPases, transporters that are activated by the phytotoxin fusicoccin, appear to be regulated in a similar fashion.

Two cDNA clones encoding 14-3-3 homologs from tomato fruit.

Two tomato cDNA clones with homology to the 14-3-3 family of proteins were identified through immunoscreening a ripening tomato fruit library (Clontech). These two clones share approx. 71% identity at the nucleotide level and 84% identity at the deduced amino acid level, with radical amino acid substitutions clustering at the acidic carboxy-terminus. Southern hybridization data indicate that each clone represents a unique gene. Distinct transcript accumulation patterns during tomato fruit ripening together with the homology to brain regulatory proteins suggest potential involvement in fruit development.

Plant 14-3-3s: omnipotent metabolic phosphopartners?

The accurate regulation of metabolism is crucial to the existence all organisms. The inappropriate activation of metabolic enzymes can waste precious energy; likewise, the failure to activate metabolic enzymes can disrupt homeostasis and lead to suboptimal cellular (and organismic) responses. Plants use several means to control their metabolic proteins, including a two-step process of protein phosphorylation and subsequent binding by phosphospecific binding proteins termed 14-3-3 proteins. Sehnke and Ferl discuss how 14-3-3 proteins regulate the activity of nitrate reductase and the H(+)-ATPase pump in plants, and compare the functions of 14-3-3 proteins in plants and animals.

Nucleotide sequence of an actin gene from Arabidopsis thaliana.

An 1830-bp genomic DNA segment containing an Arabidopsis thaliana actin gene, AAc1, has been cloned and sequenced. The AAc1 gene is present as a single-copy gene, but at least two other actin-like genes have been detected. Comparison of the nucleotide sequence of AAc1 with other cloned plant actin genes reveals four exons separated by three introns conservatively located in all plant actin genes. The deduced amino acid sequence has also been compared with actin protein sequences from other plants.

14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases.

Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: omega> chi> upsilon>>> phi, psi.

Four Arabidopsis thaliana 14-3-3 protein isoforms can complement the lethal yeast bmh1 bmh2 double disruption.

The 14-3-3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction. Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14-3-3 proteins; one complemented very poorly and one did not complement. However, the expression of the latter two isoforms was very low. These results show that at least four of the six A. thaliana isoforms are able to perform the same function(s) as the yeast 14-3-3 proteins.

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Ultrastructural correlates of anaerobic stress in corn roots.

Cytological events in Zea mays root meristem were followed during 26 hr of anaerobic treatment. From 8 to 14 hr, mitochondria swelled drastically, Golgi apparatus actively produced vesicles, endoplasmic reticulum proliferated, and chromatin strongly condensed. Plastids appeared normal. By 26 hr, however, Golgi activity receded, mitochondria assumed long, polymorphic shapes, chromatin partly dispersed, and plastids swelled. ER remained prominent, and the cytoplasm contained long fibrous inclusions. This preliminary study emphasizes the need to examine quantitatively all cellular organelles periodically for longer periods when following events of stress or pathology. Our observations corroborate scattered reports in the literature for single organelles under anaerobic stress and represent the first set of correlated observations on the entire spectrum of cellular events.

Effects of a spaceflight environment on heritable changes in wheat gene expression.

Once it was established that the spaceflight environment was not a drastic impediment to plant growth, a remaining space biology question was whether long-term spaceflight exposure could cause changes in subsequent generations, even if they were returned to a normal Earth environment. In this study, we used a genomic approach to address this question. We tested whether changes in gene expression patterns occur in wheat plants that are several generations removed from growth in space, compared to wheat plants with no spaceflight exposure in their lineage. Wheat flown on Mir for 167 days in 1991 formed viable seeds back on Earth. These seeds were grown on the ground for three additional generations. Gene expression of fourth-generation Mir flight leaves was compared to that of the control leaves by using custom-made wheat microarrays. The data were evaluated using analysis of variance, and transcript abundance of each gene was contrasted among samples with t-tests. After corrections were made for multiple tests, none of the wheat genes represented on the microarrays showed a statistically significant difference in expression between wheat that has spaceflight exposure in their lineage and plants with no spaceflight exposure. This suggests that exposure to the spaceflight environment in low Earth orbit space stations does not cause significant, heritable changes in gene expression patterns in plants.

Microgravity effects on leaf morphology, cell structure, carbon metabolism and mRNA expression of dwarf wheat.

The use of higher plants as the basis for a biological life support system that regenerates the atmosphere, purifies water, and produces food has been proposed for long duration space missions. The objective of these experiments was to determine what effects microgravity (microg) had on chloroplast development, carbohydrate metabolism and gene expression in developing leaves of Triticum aestivum L. cv. USU Apogee. Gravity naive wheat plants were sampled from a series of seven 21-day experiments conducted during Increment IV of the International Space Station. These samples were fixed in either 3% glutaraldehyde or RNAlater or frozen at -25 degrees C for subsequent analysis. In addition, leaf samples were collected from 24- and 14-day-old plants during the mission that were returned to Earth for analysis. Plants grown under identical light, temperature, relative humidity, photoperiod, CO(2), and planting density were used as ground controls. At the morphological level, there was little difference in the development of cells of wheat under microg conditions. Leaves developed in mug have thinner cross-sectional area than the 1g grown plants. Ultrastructurally, the chloroplasts of microg grown plants were more ovoid than those developed at 1g, and the thylakoid membranes had a trend to greater packing density. No differences were observed in the starch, soluble sugar, or lignin content of the leaves grown in microg or 1g conditions. Furthermore, no differences in gene expression were detected leaf samples collected at microg from 24-day-old leaves, suggesting that the spaceflight environment had minimal impact on wheat metabolism.

Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis.

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.

ARF-B(2): A Protein Complex that Specifically Binds to Part of the Anaerobic Response Element of Maize Adh 1.

Crude whole cell extracts from maize (Zea mays L.) suspension cells were examined for DNA binding proteins that specifically interact with a portion of the maize Adh 1 promoter that was previously shown to be in contact with a trans-acting factor in vivo. A 17 base pair, double-stranded oligonucleotide probe was constructed that centered around a strong in vivo dimethylsulfate footprint (B(2)) that coincides with part of the anaerobic response element (ARE). Gel retardation assays were used to characterize a major, specific DNA binding protein activity found in the crude extracts. The activity is present in both aerobic and hypoxically treated cultures and has been designated ARF-B(2) (ARE binding factor). ARF-B(2) appears to be a multicomponent complex, with a 54 kilodalton subunit termed ARF-B(2)alpha in primary contact with the target DNA.

In vivo footprinting reveals unique cis-elements and different modes of hypoxic induction in maize Adh1 and Adh2.

The transcriptional activation of maize alcohol dehydrogenase-1 (Adh1) and alcohol dehydrogenase-2 (Adh2) is accompanied by changes in the chromatin structure within the 5'-flanking region of each gene. The positions of DNA-binding factors bound to the 5'-flanking regions were determined by in vivo dimethyl sulfate footprinting of maize suspension cultures over 8 hours of hypoxic induction. In Adh1 there are two types of DNA-binding factors associated with the promoter region. One set of factors is constitutively associated with the cis-regulatory anaerobic response element, whereas two additional factors bind only after Adh1 has been induced by hypoxic stress. Returning hypoxically stressed cells to an aerobic environment restores the dimethyl sulfate footprint observed for the uninduced Adh1 gene. In contrast, all of the factors bound to the 5'-flanking region of Adh2 are constitutively present and unchanged by hypoxia. There is one footprint site common to both Adh1 and Adh2, but it is not an anaerobic response-like element.

In vivo detection of regulatory factor binding sites in the 5' flanking region of maize Adh1.

In vivo dimethyl sulfate footprinting experiments have been used to locate the binding sites of regulatory molecules in the TATA proximal portion of the maize alcohol dehydrogenase-1 gene. In several tissues and organs, Adh1 is transcriptionally induced by anaerobic stress, and the various alleles of Adh1 can show a quantitative differential response to induction. Two regulatory molecules were found to be bound to the promoter only when the gene was induced. The binding sites for these factors mapped to regions located at -100 to -108 and -186 to -190 relative to the site of transcript initiation. Two additional regulatory molecules were bound to sites located at positions -117 to -120 and -138 to -145 regardless of the state of transcription. However, the factor bound at the -138 to -145 region was found to alter its binding characteristics when the gene was induced.

Characterization of the Arabidopsis Adh G-box binding factor.

Protein-DNA interaction at an inverted repeat of the sequence 5'-GTGG-3' (G-box) has been associated with the transcription of several plant genes [Giuliano, G., et al. (1988). Proc. Natl. Acad. Sci. USA 85, 7089-7093; Ferl, R.J., and Laughner, B.H. (1989). Plant Mol. Biol. 12, 357-366; Schulze-Lefert, P., et al. (1989). EMBO J. 8, 651-656]. We characterized the binding of the Arabidopsis G-box binding factor (GBF) from whole-cell extracts and fractionated extracts to the G-box of alcohol dehydrogenase (Adh) using gel mobility shift assays. DNase I footprinting localized the region of GBF/G-box interaction to two sites, one apparent high-affinity binding site (-227 to -201) and a possible low-affinity binding site (-193 to -182). DNA-protein cross-linking demonstrated that the G-box is bound by proteins of two sizes, 31 kilodaltons and 18 kilodaltons. In addition, we found that in vitro the interaction of GBF from Arabidopsis suspension cultures or leaves with the Adh G-box is indistinguishable, and that there is evidence of multiple protein-protein interactions.

The complete nucleotide sequence of the small-subunit ribosomal RNA coding region for the cycad Zamia pumila: phylogenetic implications.

The DNA sequence of the small-subunit ribosomal RNA coding region for the cycad Zamia pumila L. was determined. The Zamia small-subunit rRNA was found to be 1813 nucleotides in length and approximately 92% identical to published angiosperm small-subunit rRNA sequences. Conserved regions interspersed with variable regions are observed corresponding to those found in other eukaryotic small-subunit sequences. Using representatives from protist, fungal, plant, and animal groups, a distance matrix was constructed of average nucleotide substitution rates for pairs of organisms. Phylogenetic trees were inferred from similarities between sequences. The sequence of Zamia represents the earliest divergence from the higher plant lineage reported to date for small-subunit rRNA data. Inferred phylogenies also support a monophyletic origin for the angiosperms consistent with studies citing phenotypic characters.

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo.

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from -44 to -79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.

Functional elements of the Arabidopsis Adh promoter include the G-box.

The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5' to the transcription start site, and deletion to -177 results in greater than 90% reduction in promoter activity. Site-specific mutations of G-box bases result in greater than 60% reduction in activity and disrupt G-box factor binding in vitro.