Preparation of transcriptionally active nuclei from etiolated Arabidopsis thaliana.

Despite its emergence as the plant model system, there are few reports that describe protocols for the isolation of functional nuclei from Arabidopsis thaliana and their use in nuclear run-on assays or in preparation of nuclear extracts. This is especially true for etiolated seedlings. Here we report conditions, optimized for use in Arabidopsis, which allow for the isolation of enriched fractions of functional nuclei from less than 2 g of etiolated or light-grown tissue. The nuclei are capable of incorporating 3H-UTP into TCA-insoluble RNA, and also incorporate 32P-CTP into transcripts that can subsequently be hybridized to specific filter-bound DNA target sequences. The functional nuclei are sensitive to the transcriptional inhibitors actinomycin D and α-amanitin, confirming that the transcription observed is both template dependent and relies on nuclear polymerase II.

Cloning and characterisation of PGA1 and PGA2: two G protein alpha-subunits from pea that promote growth in the yeast Saccharomyces cerevisiae.

We report here on the cloning and characterization of two G protein alpha-subunits from pea: PGA1 and PGA2. Based on DNA gel blot analysis, PGA1 and PGA2 are the only Galpha homologous sequences in pea. RT-PCR analysis reveals that PGA1 and PGA2 transcripts are present in a variety of adult pea tissues. However, PGA2 mRNA is consistently detected at a lower level than PGA1 and demonstrates some degree of tissue specificity relative to PGA1. In the apical bud of pea seedlings, PGA1 and PGA2 transcripts decrease in response to 24 h of white light following growth for 6 days in darkness. The G protein mediated, yeast mating pathway was used to analyse the function of PGA1 and PGA2 in vivo. PGA1 downregulates the mating pathway, but through a mechanism that is independent of Gbetagamma sequestration. Unexpectedly, both PGA1 and PGA2 promote growth through a mating pathway independent mechanism.

Blue light-directed destabilization of the pea Lhcb1*4 transcript depends on sequences within the 5' untranslated region.

Pea seedlings grown in continuous red light accumulate significant levels of Lhcb1 RNA. When treated with a single pulse of blue light with a total fluence >10(4) micromol m(-2), the rate of Lhcb1 transcription is increased, whereas the level of Lhcb1 RNA is unchanged from that in control seedlings. This RNA destabilization response occurs in developing leaves but not in the apical bud. The data presented here indicate that the same response occurs in the cotyledons of etiolated Arabidopsis seedlings. The blue light-induced destabilization response persists in long hypocotyl hy4 and phytochrome phyA, phyB, and hy1 mutants as well as in far-red light-grown seedlings, indicating that neither CRY1 (encoded by the hy4 locus) nor phytochrome is the sole photoreceptor. Studies with transgenic plants indicate that the destabilization element in the pea Lhcb1*4 transcript resides completely in the 5' untranslated region.

Regions of the pea Lhcb1*4 promoter necessary for blue-light regulation in transgenic Arabidopsis.

Pea (Pisum sativum) and Arabidopsis contain similar, if not identical, blue-light (BL)-responsive systems that alter expression of specific members of the Lhcb (light-harvesting chlorophyll-binding) gene family. In both plants a single, short pulse of low-fluence BL (threshold = 10(-1) micromol m-2) causes an increase in the rate of transcription from specific members of the Lhcb gene family in etiolated seedlings. Constructs of the BL-regulated pea Lhcb1*4 promoter (PsLhcb1*4) were created, which altered sequences previously implicated in light responses, deleted the 5'-promoter sequence, or removed the 5'-untranslated region. These constructs were tested for BL induction in transgenic Arabidopsis. The PsLhcb1*4 promoter deletions to -150 bp maintained normal fluence response, time course, and reciprocity characteristics. The 5'- untranslated region contained enhancer elements, but was not necessary for BL induction. The -95 to +2 promoter was capable of responding to BL, whereas sequences from -50 were not. Promoters that lack conserved light-regulatory elements or sequences directly implicated in phytochrome and circadian responses retained BL activity, suggesting that the low-fluence BL response utilizes regions of the promoter independent of those that modulate the phytochrome and circadian responses.

Microhabitat use, trophic patterns, and the evolution of brain structure in African cichlids.

The species assemblages of cichlids in the three largest African Great Lakes are among the richest concentrations of vertebrate species on earth. The faunas are broadly similar in terms of trophic diversity, species richness, rates of endemism, and taxonomic composition, yet they are historically independent of each other. Hence, they offer a true and unique evolutionary experiment to test hypotheses concerning the mutual dependencies of ecology and brain morphology. We examined the brains of 189 species of cichlids from the three large lakes: Victoria, Tanganyika, and Malawi. A first paper demonstrated that patterns of evolutionary change in cichlid brain morphology are similar across taxonomic boundaries as well as across the three lakes [van Staaden et al., 1995 ZACS 98: 165-178]. Here we report a close relationship between the relative sizes of various brain structures and variables related to the utilization of habitat and prey. Causality is difficult to assign in this context, nonetheless, prey size and agility, turbidity levels, depth, and substrate complexity are all highly predictive of variation in brain structure. Areas associated with primary sensory functions such as vision and taste relate significantly to differences in feeding habits. Turbidity and depth are closely associated with differences in eye size, and large eyes are associated with species that pick plankton from the water column. Piscivorous taxa and others that utilize motile prey are characterized by a well developed optic tectum and a large cerebellum compared to species that prey on molluscs or plants. Structures relating to taste are well developed in species feeding on benthos over muddy or sandy substrates. The data militated against the existence of compensatory changes in brain structure. Thus enhanced development of a particular function is generally not accompanied by a parallel reduction of structures related to other modalities. Although genetic and environmental influences during ontogeny of the brain cannot be isolated, this study provides a rich source of hypotheses concerning the way the nervous system functions under various environmental conditions and how it has responded to natural selection.

Correct blue-light regulation of pea Lhcb genes in an Arabidopsis background.

Irradiation of etiolated Arabidopsis or pea, or dim-red-light-grown pea seedling with a single, short (under 10 s) pulse of blue light (threshold at 0.1 mumol/m2) is sufficient to induce the expression of specific members of the Lhcb gene family including the pea Lhcb1*4 gene and the Arabidopsis Lhcb1*3 gene. Other Lhcb genes, such as the pea Lhcb1*3 gene and the Arabidopsis Lhcb1*1 and 1*2 genes are unaffected by this blue-light treatment. Transgenic Arabidopsis bearing pea Lhcb1*3::Gus (beta-glucuronidase), pea Lhcb1*4::Gus or Arabidopsis Lhcb1*3::Gus constructs were used to determine if pea and Arabidopsis employ a similar mechanism to achieve blue-light induced Lhcb expression. Examination of the respective Gus expression patterns in white-light-grown seedlings indicates that the pea promoters are active and properly expressed in the Arabidopsis background. Irradiation of dark-grown Arabidopsis with a 20 s pulse of blue light with a total fluence of 100 mumol/m-2 results in expression of the pea Lhcb1*4::Gus (beta-glucuronidase) construct, but not of the pea Lhcb1*3::Gus construct indicating that the pea promoters respond correctly to blue light in the Arabidopsis background. Fluence-response, time-course and reciprocity characteristics for the blue-light-induced expression of the pea Lhcb1*4::Gus construct closely resemble those of the endogenous Arabidopsis Lhcb genes, confirming the proper interpretation of the Arabidopsis blue-light-signaling mechanism by the pea Lhcb1*4 promoter and suggesting that the signaling mechanisms in the two plants are very similar, if not identical. Fluence response data for the steady-state level of transcript derived from an Arabidopsis Lhcb1*3::Gus construct extending 200 bp upstream of the site of transcription indicate that the blue light responsive elements(s) are contained within this 200 bp region.

Individual Members of the Cab Gene Family Differ Widely in Fluence Response.

Chlorophyll a/b-binding protein genes (Cab genes) can be extremely sensitive to light. Transcript accumulation following a red light pulse increases with fluence over 8 orders of magnitude (L.S. Kaufman, W.F. Thompson, W.R. Briggs [1984] Science 226: 1447-1449). We have constructed fluence-response curves for individual Cab genes. At least two Cab genes (Cab-8 and AB96) show a very low fluence response to a single red light pulse. In contrast, two other Cab genes (AB80 and AB66) fail to produce detectable transcript following a single pulse of either red or blue light but are expressed in continuous red light. Thus, very low fluence responses and high irradiance responses occur in the same gene family.

A 13-week toxicologic and pathologic evaluation of prolonged cytochromes P450 inhibition by 1-aminobenzotriazole in male rats.

The pharmacologic, toxicologic, and microscopic effects of 100 mg/kg/day of 1-Aminobenzotriazole (ABT), a suicide substrate inhibitor of cytochromes P450, were assessed in male Sprague-Dawley rats over a 13-week period. Hepatic cytochromes P450 levels and resorufin dealkylase activity were decreased to less than 30% of control values beginning at Day 2 and from Day 8 to Day 92. These decreases were not accompanied by overt clinical toxicity, e.g., changes in body weight, food consumption, or clinical appearance, during the study. Hemoglobin, hematocrit, and erythrocyte counts were slightly decreased at 8, 29, and 92 days and were accompanied by increased spleen weights and extramedullary hematopoiesis. Additionally, mean corpuscular hemoglobin concentration, mean corpuscular volume, red cell distribution width, and mean corpuscular hemoglobin were slightly increased at 92 days. Increases in liver weights at 8, 29, and 92 days were accompanied by centrilobular hypertrophy and intracytoplasmic vacuolization consistent with lipid accumulation. Thyroid stimulating hormone (TSH) was slightly elevated and triiodothyronine and thyroxine were slightly decreased at 29 days. TSH was also slightly elevated at 8 and 92 days, and thyroid gland weights were increased at 8, 29, and 92 days with microscopic evidence of hyperplasia and hypertrophy of thyroid gland follicular cells. Increased adrenal weights and hypertrophy of the zona fascicularis of the adrenal gland were observed at 8, 29, and 92 days. Kidney weights were also increased at these assessments. Changes in the thyroid gland, the thyroid hormone profile, and the liver may reflect increased synthesis of microsomal enzymes, an effect that is sometimes difficult to demonstrate directly with suicide substrate inhibitors of cytochromes P450. In general, the effects of daily ABT administration to male rats at a dose that significantly reduces oxidative metabolism over a 13-week period were considered to be well-tolerated under controlled laboratory conditions.

Blue-Light Regulation of the Arabidopsis thaliana Cab1 Gene.

The steady-state level of Cab RNA in etiolated Arabidopsis thaliana increases as a result of a single pulse of blue light. The threshold for the response is at or below 10[deg] [mu]mol m-2 and begins within 1 h of irradiation. The response is not prevented by far-red treatment, and the blue-light source used does not elicit and observable very low fluence phytochrome response for RbcS RNA. The time course for blue-light-induced transcript accumulation differs from that of red, the blue beginning more quickly. Transcripts derived from the Cab1 (AB140; Lhcb1*3) member of the gene family are responsible in part for the blue-light-induced accumulation. This is the same member of the gene family that is responsible for phytochrome-induced Cab gene expression (G.A. Karlin-Neumann, L. Sun, E.M. Tobin [1988] Plant Physiol 88: 1323-1331). The mutant hy4, which lacks blue-light-induced suppression of hypocotyl elongation, retains the ability of Cab RNA to respond to blue light.

A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas.

Heterotrimeric GTP-binding regulatory proteins (G proteins) have been identified as part of signal transduction systems in a wide variety of organisms. In this paper, we establish the presence of a G protein associated with the plasma membranes of the apical bud of etiolated peas. The GTPase activity is induced by low fluences of blue light administered to plasma membrane-enriched fractions. The activity is not responsive to red-light irradiation and is specific for GTP. The threshold for the excitation of the GTPase activity in vitro is less than 10(-1) mumol.m-2 of blue light, consistent with participation in the blue low-fluence system identified in the same tissue. A 40-kDa polypeptide is recognized by polyclonal antisera directed against the alpha subunit of the G protein transducin. The polypeptide also serves as a substrate for ADP-ribosylation by cholera and pertussis toxins. The ability of the 40-kDa polypeptide to serve as substrate for the toxin-mediated ribosylation is mediated by blue-light irradiation, implying that the 40-kDa polypeptide is the alpha subunit of a blue-light-stimulated G protein. The 40-kDa polypeptide binds a nonhydrolyzable photoaffinity-labeling analog of GTP only after irradiation with blue light. The protein we have described may function as an alpha subunit of a G protein active in the process of light-mediated development in higher plants.

Rapid transcriptional regulation of the Cab and pEA207 gene families in peas by blue light in the absence of cytoplasmic protein synthesis.

We have analyzed the fluence-response and time-course characteristics, and the requirements for protein synthesis, for the blue-light (BL) regulated transcription of the nuclear-coded Cab, pEA207, and pEA25 gene families in pea (Pisum sativum L.). Fluence-response curves indicate two BL responses: a blue low-fluence (BLF) response with a threshold at or below 10(-1) µmol·m(-2) of BL and a blue high-fluence (BHF) response with a threshold between 10(1) and 10(3) µmol·m(-2) of BL. Excitation of the photomorphogenic system responsible for the BLF response results in increased Cab, and decreased pEA25, transcription. Excitation of the photomorphogenic system responsible for the BHF response results in decreased pEA207 transcription and induction of turnover for Cab RNA and pEA207 RNA. Altered rates of transcription for the Cab and pEA207 gene families are apparent within 15 min of BL treatment and remain in effect for at least 24 h. The effect of BL on pEA25 transcription is not apparent until 3-5 h after the BL treatment. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, has no effect on the altered rates of pEA207 and Cab transcription. We conclude from these results that the BLF response for Cab transcription and the BHF response for pEA207 transcription probably occurs without the expression of intervening regulatory genes coded within the nucleus or the translation of pre-existing transcripts derived from nuclear-coded genes.

Structural analysis of the short length ribosomal DNA variant from Pisum sativum L. cv. Alaska.

The genomic clone, RRNpss1, representing the short ribosomal DNA length variant in Pisum sativum L. cv. Alaska, has been isolated and the 2859 bp intergenic spacer, along with the 25S rRNA 3' border and 18S rRNA 5' border, has been sequenced. The intergenic spacer contains nine tandem repeats, approximately 180 bp in length, which show greater than 80% sequence homology to each other. The RNA polymerase I transcription start site and a processing site, located 776 bp and 536 bp upstream of the 5' end of 18S rRNA, respectively, have been determined using S1 analysis. The region surrounding the +1 site shows strong homology between the positions -6 to +10 to the rDNA sites of initiation in radish, maize, and wheat. The sequence CATGCAAA is located 19 bp upstream of the site of initiation, and appears once within each subrepeat and twice more between the end of the subrepeat array and the site of initiation. A previously characterized HpaII site which shows developmental regulation of methylation is located 31 bp downstream of the site of initiation. Using RFLP linkage analysis, the short rDNA length variant of cv. Alaska is assigned to Chromosome 4 where it is genetically independent of the long rDNA length variant which is putatively assigned to Chromosome 7.

Increased rRNA gene activity during a specific window of early pea leaf development.

rRNA gene transcription rates were determined during light-mediated leaf development in Pisum sativum. The rate of transcription was observed to increase within 1 day of exposure to light and return to control levels 4 days after exposure. A striking similarity was observed between periods of elevated rRNA gene transcription and increased mitotic activity, suggesting a possible link between the two events.

Two distinct blue-light responses regulate epicotyl elongation in pea.

Blue light induces a long-term suppression of epicotyl elongation in red-light-grown pea (Pisum sativum L.) seedlings. The fluence-response characteristics are bell-shaped, indicating the possibility of two different blue-light responses: a lower fluence response causing suppression and a higher fluence response alleviating the suppression. To determine if two responses are in effect, we have grown pea seedlings under dark conditions hoping to eliminate one or the other response. Under these growth conditions, only the lower fluence portion of the response (suppression of elongation) is apparent. The kinetics of suppression are similar to those observed for the lower fluence response of red-light-grown seedlings. The response to blue light in the dark-grown seedlings is not due to the excitation of phytochrome because a pulse of far-red light large enough to negate phytochrome-induced suppression has no effect on the blue-light-induced suppression. Furthermore, treatment of the dark-grown seedlings with red light immediately prior to treatment with high fluence blue light does not elicit the higher fluence response, indicating that the role of red light in the blue high fluence response is to allow the plant to achieve a specific developmental state in which it is competent to respond to the higher fluences of blue light.

Two distinct blue-light responses regulate the levels of transcripts of specific nuclear-coded genes in pea.

Fluence-response characteristics for bluelight(BL)-mediated changes in the steady-state levels ofCab-, pEA215 and pEA207-RNA in red-light(RL) grown pea (Pisum sativum L. cv. Alaska) seedlings indicate the existence of two BL responses: a blue-lowfluence (BLF) response, causing an increase inCab- and pEA215-RNA, and a blue-high-fluence (BHF) response, causing a return to control levels forCab- and pEA215-RNA and a decrease in pEA207-RNA levels (Warpeha and Kaufman, 1989, Plant Physiol.,91, 1030-1035). We now show that under dark growth conditions, only the BLF response is apparent;Cab- and pEA215-RNA increase at all fluences tested, whereas pEA207-RNA levels are unaltered over the range of BL fluence tested. The treatment of dark-grown seedlings with RL immediately prior to BHF irradiation does not elicit the BHF response forCab-, pEA215 and pEA207-RNA, indicating that the role of growth in RL is to enable the seedling to reach a particular developmental state, rather than ensuring the presence of active phytochrome at the time of BL-irradiation. The apical bud of RL-grown seedlings has only the BLF response;Cab-RNA levels increase while pEA207-RNA exhibits no change at any of the fluences tested. The developing leaves of the fourth node show the BHF response; bothCab- and pEA207-RNA decrease following treatment with high-fluence BL. These data also indicate the necessity for reaching a specific developmental state before the BHF response can be activated.

Blue-Light Regulation of Specific Transcript Levels in Pisum sativum: Fluence-Response, Time-Course, and Reciprocity Characteristics.

The expression of many nuclear genes in plants is light regulated. We have examined the fluence-response, time-course, and reciprocity characteristics of four nuclear, blue-light-regulated transcripts in Pisum sativum L. var Alaska: Cab RNA, pEA207 RNA, pEA215 RNA, and pEA25 RNA. To avoid complications due to blue-light-induced transformation of phytochrome, we have adapted the procedure of measuring blue-light-induced changes in steady-state-RNA levels in seedlings grown in continuous red light. The fluence-response curves for Cab and pEA215 RNA are bell-shaped, with peak accumulations at 10(2) and 10(1) micromoles per square meter, respectively. No response is observed at 10(4) micromoles per square meter. pEA25 RNA has threshold and saturation fluences below 10(-1) micromoles per square meter. pEA207 RNA has a threshold at 10(2) micromoles per square meter and saturates above 10(4) micromoles per square meter. pEA215 and Cab RNA start to increase within 1 hour after the 10(1) micromoles per square meter pulse, and finish accumulation by 5 hours. The decrease in pEA207 RNA in response to 10(4) micromoles per square meter and pEA25 RNA in response to 10(1) or 10(4) micromoles per square meter starts between 3 and 5 hours after the blue-light pulse. The Bunsen-Roscoe Law of Reciprocity is followed in the Cab, pEE215, pEA25, and pEA207 RNA responses to 10(1) and 10(4) micromoles per square meter.

Blue-light regulation of transcription for nuclear genes in pea.

The expression of many nuclear genes in plants is light regulated. Previous investigations have shown that the excitation of phytochrome can affect transcription rate and steady-state RNA levels for several of these genes. No direct demonstration of the effects of blue light has been reported. We have identified several nuclear genes in pea whose transcription rate and steady-state RNA levels are affected by a single pulse of blue light. Pea seedlings, grown in continuous red light to saturate any phytochrome response, were treated with a single pulse (10(3) mumol.m(-2)) of blue light 6 days after planting. The blue-light treatment resulted in an increase in the steady-state RNA level and rate of transcription for the Cab (chlorophyll a/b binding protein) gene family and a decrease in the steady-state RNA level and the rate of transcription for the gene families corresponding to cDNA clones pEA25 and pEA207. Changes in the rate of transcription for Cab and pEA207 are apparent within 3 hr of the blue-light treatment.

Blue-Light Regulation of Epicotyl Elongation in Pisum sativum.

Blue light is known to induce suppression of stem elongation. To avoid the complication of blue-light-induced transformation of phytochrome we have adapted the procedure of measuring blue-light-induced suppression of stem elongation in Pisum sativum L. var Alaska grown under continuous red light. The resulting fluence-response curve for suppression of epicotyl elongation measured twenty-four hours after a blue-light treatment is bell-shaped, with the peak of suppression between 10(0) and 10(1) micromoles per square meter, and no suppression at 10(4) micromoles per square meter. Suppression is first observed 5 and 11 hours after the blue-light treatment for the fourth and third internodes, respectively. No significant differences in elongation rates were noted for the 10(4) micromoles per square meter treated seedlings throughout the 24 hour period. Reciprocity holds for both third and fourth internodes in response to 10(1) and 10(4) micromoles per square meter of blue light over the range of irradiation times tested (10(0) to 10(4) seconds, 10(1) micromoles per square meter; 10(0) to 10(3) seconds, 10(4) micromoles per square meter). In contrast to the bell-shaped fluence-response obtained for epicotyl elongation, measurements of chlorophyll and carotenoid accumulation indicate increasing accumulation with increasing fluence.

Cholinergic mechanisms of lordotic behavior in rats.

Cholinergic mechanisms of lordotic behavior were studied in hooded rats using behavioral techniques and autoradiographic analysis of the diffusion of [3H]N methyl scopolamine ([3H]NMS) applied to the hypothalamus. Bilateral cannulae were implanted chronically in the region of the ventromedial nuclei (VMN). A series of cholinergic drugs and estradiol (E2) were administered to each rat during successively induced periods of estrus. Robust inhibition of lordosis resulted from administration of atropine and scopolamine. Robust facilitation followed carbachol administration. Pirenzepine, hexamethonium, and tetraethylammonium did not inhibit lordosis. [3H]NMS, the last drug in the series to be administered, inhibited lordosis in two of three rats. Autoradiographic analysis of [3H]NMS diffusion in these rats revealed that radioactivity was confined entirely to the hypothalamus and appeared in the region of the VMN. In an additional experiment, the in vitro binding characteristics of [3H]NMS in the basomedial hypothalamus were examined. The VMN appeared lightly labeled and were surrounded by regions of darker labeling. We conclude that cholinergic drugs influence lordotic behavior when applied in crystalline form in the vicinity of the VMN of the female rat hypothalamus.