Use of alpha-, beta-Estrogen Receptor as a "new tool" for detection of specific small molecule activity.

Cell-based screening methods for nuclear receptor ligands that use transgenic plant cells expressing a single human NR may have advantages over other eukaryotic systems which express multiple NRs. For example, signal-to-noise ratio might be improved because ligands would be less likely to bind to other NRs and/or less likely to cause confounding functional changes in plant cells. As a first step toward this aim we have expressed in plants truncated human estrogen receptor (ER) constructs linked to reporters, or selective markers such as luciferase, green fluorescent protein (GFP) and hygromycin. A variety of ligands for the ER (including estradiol and known phytoestrogens) have then been tested for their ability to over-express the linked marker gene(s) which could be measured (luciferase activity), visualized under fluorescent microscopy (GFP activity), or selected on antibiotic-containing media (Hygromycin B). Our results show a close association between the effects of ER ligands in the transgenic plant roots and their effects on native ERs in mammalian cells. With the stable expression of an ERalpha-GFP ligand detection system in A. thaliana, the estradiol- mediated response in transgenic roots is inhibited by an ER partial agonist (tamoxifen) and an antagonist (fulvestrant) at concentrations relevant to their use in breast cancer. We conclude that it is possible to express human NRs in plants in a form that can report on exogenous or endogenous ER ligands and that these constructs have a pharmacology which is relevant to ligands for the native NRs in human cells.

Improved growth and stress tolerance in the Arabidopsis oxt1 mutant triggered by altered adenine metabolism.

Plants perceive and respond to environmental stresses with complex mechanisms that are often associated with the activation of antioxidant defenses. A genetic screen aimed at isolating oxidative stress-tolerant lines of Arabidopsis thaliana has identified oxt1, a line that exhibits improved tolerance to oxidative stress and elevated temperature but displays no apparent deleterious growth effects under non-stress conditions. Oxt1 harbors a mutation that arises from the altered expression of a gene encoding adenine phosphoribosyltransferase (APT1), an enzyme that converts adenine to adenosine monophosphate (AMP), indicating a link between purine metabolism, whole-plant growth responses, and stress acclimation. The oxt1 mutation results in decreased APT1 expression that leads to reduced enzymatic activity. Correspondingly, oxt1 plants possess elevated levels of adenine. Decreased APT enzyme activity directly correlates with stress resistance in transgenic lines that ectopically express APT1. The metabolic alteration in oxt1 plants also alters the expression of several antioxidant defense genes and the response of these genes to oxidative challenge. Finally, it is shown that manipulation of adenine levels can induce stress tolerance to wild-type plants. Collectively, these results show that alterations in cellular adenine levels can trigger stress tolerance and improve growth, leading to increases in plant biomass. The results also suggest that adenine might play a part in the signals that modulate responses to abiotic stress and plant growth.

Reactive oxygen species regulate alkaloid metabolism in undifferentiated N. tabacum cells.

Plants produce an immense number of natural products and undifferentiated cells from various plant tissues have long been considered an ideal source for their synthesis. However, undifferentiated plant cells often either lose their biosynthetic capacity over time or exhibit immediate repression of the required pathways once dedifferentiated. In this study, freshly prepared callus tissue was employed to further investigate the regulation of a natural product pathway in undifferentiated tobacco cells. Putrescine N-methyltransferase (PMT) is a pathway-specific enzyme required in nicotinic alkaloid production in Nicotiana species. Callus derived from transgenic Nicotiana tabacum plants harboring PMT promoter-GUS fusions were used to study factors that influence PMT expression. Under normal callus growth conditions in the presence of light and auxin, PMT promoter activity was strongly repressed. Conversely, dark conditions and the absence of auxin were found to upregulate PMT promoter activity, with light being dominant to the repressive effects of auxin. Since reactive oxygen species (ROS) are known by-products of photosynthesis and have been implicated in signaling, their involvement was investigated in transgenic callus by treatment with the ROS scavenger, dimethylthiourea, or catalase. Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, both ROS scavengers resulted in significant induction of PMT promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS impact the regulation of the alkaloid pathway in undifferentiated cells and have implications for regulation of the pathway in other plant tissues.

The A312L 5'-UTR of Chlorella virus PBCV-1 is a translational enhancer in Arabidopsis thaliana.

PBCV-1 (Paramecium bursaria Chlorella virus) is a large double stranded DNA virus that replicates in certain eukaryotic chlorella like green algae. The PBCV-1 A312L gene encodes a 33-kDa protein whose function currently is unknown. The 5'-UTR of the A312L mRNA is 153 nucleotides, longer than the 5'-UTR in any other PBCV-1 gene. The sequence 5'-AAAC was repeated 17 times within 156bp 5' to the A312L gene start codon and this sequence was repeated 13 times continuously in the 5'-UTR of the mRNA. Recombinant genes were constructed in vector pBI121 that contained the A312L 5'-UTR, in both the forward and inverse-complement orientations, fused to the GUS gene under the control of the CaMV 35S promoter. These constructs were introduced into Arabidopsis thaliana and the results indicated that the A312L 5'-UTR functions as a translational enhancer only in the forward orientation. Overall, the ratio of GUS enzyme activity to GUS mRNA was 15-fold higher in constructs derived from the A312L 5'-UTR in the forward orientation as compared to constructs containing the 5'-UTR in the inverse-complement orientation or those lacking the A312L 5'-UTR.

S-adenosylmethionine mediates glutathione efficacy by increasing glutathione S-transferase activity: implications for S-adenosyl methionine as a neuroprotective dietary supplement.

When maintained on a folate-deficient, iron-rich diet, transgenic mice lacking in apolipoprotein E (ApoE-/- mice) demonstrate impaired activity of glutathione S-transferase (GST), resulting in increased oxidative species within brain tissue despite abnormally high levels of glutathione. These mice also exhibit reduced levels of S-adenosyl methionine (SAM) and increased levels of its hydrolysis product S-adenosyl homocysteine, which inhibits SAM usage. Supplementation of the above diet with SAM restored GST activity and eliminated reactive oxygen species at the expense of stockpiled glutathione, suggesting that one or more SAM-dependent reactions were required to maintain GST activity. We examined herein the impact of SAM on GST activity using a cell-free assay. SAM stimulated GST activity in a dose-response manner when added to homogenates derived from the above ApoE-/- mice. SAM also increased activity of purified rat liver GST and recombinant GST. Filtering of SAM through a 4 kDa cutoff and systematic withholding of reaction components eliminated the possibility of any additional contaminating enzyme. These findings confirm that SAM can exert a direct effect on GST activity. Since Alzheimer's disease is accompanied by reduced GST activity, diminished SAM and increased SAH, these findings underscore the critical role of SAM in maintenance of neuronal health.

A polyadenylation factor subunit implicated in regulating oxidative signaling in Arabidopsis thaliana.

BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6. METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin-related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant. CONCLUSIONS/SIGNIFICANCE: These results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress.

Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit.

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.

Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation.

Plants are potentially important for novel therapeutic drug leads, but the slowness of conventional methods for investigation of plants limits enthusiasm in the pharmaceutical industry. To overcome some of the drawbacks, we have applied high throughput pharmacological screening (HTPS) to crude plant extracts. Using a "differential smart screen", (DSS) the spectrum of activity contained in a crude extract is measured at several closely related receptor subtypes. This spectrum is then compared to that of known compounds. A unique spectrum suggests that the extract merits further investigation. Evaluation of species and environmental libraries of whole plants has demonstrated the value of this approach for rapid prioritization of plants for investigation. In addition, genomic and genetic manipulation of plants and plant cell cultures can increase the value of DSS. For example, the whole genomic potential of a plant species for biodiversity can be accessed by using gain of function mutations to generate a "functional genomics library" of mutant clonal cultures, and the bioactivity of these cultures tested by DSS. Clones that overproduce activity differing from the wild-type plant can be identified in this way. This "Natural Products Genomics" (NPG) strategy is limited by the massive numbers of clonal cultures that are required to cover all possible gain-of-function mutations. The rapidity and efficiency of this process can be improved by using transgenic plants expressing appropriate mammalian proteins. These may be designed to make the plant cell resemble a human cell for a specific form of toxicity. Now, "unnatural selection" of resistant mutant clones can be used to provide a sub-population potentially enriched in useful compounds. Alternatively, transgenic plant cells can be used for "in situ screening" in which a mammalian receptor protein, linked to a reporter construct, such as green fluorescent protein, is expressed. Clonal cultures that produce ligands for this receptor can now be rapidly identified visually in an ultra-HTPS. Overall, our aim is to use pharmacological screening, together with functional genomic approaches, to make plant drug discovery competitive with combinatorial chemistry.

Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition.

BACKGROUND: A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition. RESULTS: In agreement with previous studies in other species, the level of unsaturation of membrane fatty acids in Arabidopsis was inversely correlated with growth temperature. The time required for the membrane fatty acids to attain the composition observed at elevated temperature was consistent with the timing required for the synthesis of new fatty acids. Comparisons of temperature-induced fatty acid alterations in membranes were made among several Arabidopsis lines including wild-type Columbia, and the compositional mutants, fad5, fad6, act1 and double mutants, fad7 fad8 and act1 fad6. The results revealed key changes that occur in response to elevated temperature regardless of the specific mutations in the glycerolipid pathway, including marked decreases in trienoic fatty acids and consistent increases in unsaturated 16:0 and in dienoic 18:2 levels. Fluorescence measurements of various mutants indicated that photosynthetic stability as well as whole plant growth at elevated temperature is influenced by certain membrane fatty acid compositions. CONCLUSIONS: The results of this study support the premise that defined proportions of saturated and unsaturated fatty acids in membrane lipids are required for photosynthetic thermostability and acclimation to elevated temperature. The results also suggest that changes in the membrane fatty acid composition brought about in response to temperature are regulated in such a way so as to achieve highly similar unsaturation levels despite mutations that alter the membrane composition prior to a high-temperature exposure. The results from examination of the mutant lines also suggest that interorganellar transfer of fatty acids are involved in mediating temperature-induced membrane alterations, and reveal steps in the fatty acid unsaturation pathway that appear to have key roles in the acclimatization of membranes to high temperature.

Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis.

Stearoyl-acyl-carrier-protein-desaturase-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is a key step, which regulates levels of unsaturated fatty acids in cells. We previously showed that stearoyl-acyl-carrier-protein-desaturase mutants ssi2/fab2 carrying a loss-of-function mutation in the plastidial glycerol-3-phosphate (G3P) acyltransferase (act1) have elevated 18:1 levels and are restored in their altered defense signaling. Because G3P is required for the acylation of 18:1 by G3P acyltransferase, it was predicted that reduction of G3P levels should increase 18:1 levels and thereby revert ssi2-triggered phenotypes. Here we show that a mutation in G3P dehydrogenase restores both salicylic acid- and jasmonic acid-mediated phenotypes of ssi2 plants. The G3P dehydrogenase gene was identified by map-based cloning of the ssi2 suppressor mutant rdc8 (gly1-3) and confirmed by epistatic analysis of ssi2 with gly1-1. Restoration of ssi2-triggered phenotypes by the gly1-3 mutation was age-dependent and correlated with the levels of 18:1. Regeneration of G3P pools by glycerol application in ssi2 and ssi2 gly1-3 plants caused a marked reduction in the 18:1 levels, which rendered these plants hypersensitive to glycerol. This hypersensitivity in ssi2 was rescued by the act1 mutation. Furthermore, overexpression of the ACT1 gene resulted in enhanced sensitivity to glycerol. Glycerol application also lowered the 18:1 content in SSI2 plants and converted these into ssi2-mimics. Our results show that 18:1 levels in plastids are regulated by means of acylation with G3P, and a balance between G3P and 18:1 is critical for the regulation of salicylic acid- and jasmonic acid-mediated signaling pathways.

Wound-induced gene expression of putrescine N-methyltransferase in leaves of Nicotiana tabacum.

Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the biosynthesis of pyrrolinium ring-containing alkaloids. Earlier studies have indicated that PMT gene expression is restricted to root tissue in Solanaceus plant species. During the analysis to further elucidate factors that govern the regulation of alkaloid synthesis, evidence was found for a novel expression pattern dictated by the 5'-flanking region of at least two members of the PMT-gene family. A 627-bp DNA fragment upstream of the NtPMT3 gene was fused to the beta-glucuronidase (GUS) reporter gene and used to produce stable transgenic lines of Nicotiana tabacum. Fluorometric and histochemical assays conducted on transgenic plants indicated high expression levels in root tissue and, in agreement with previous studies, no expression was detected in leaves. However, expression was observed in leaves when they were mechanically wounded. This expression was highly localized around the wound site and showed little evidence of long distance signaling, including lack of responsiveness to jasmonic acid. Expression was transient, with maximum levels immediately after wounding and diminishing after approximately 2-4 h. RT-PCR analysis of mRNA isolated from wild-type plants also indicated upregulation of PMT expression in leaves upon wounding as well as very low transcript levels in unwounded leaves. Low levels of PMT activity were detected in leaf tissue, which did not increase significantly upon wounding.