Different Red Light Requirements for Phytochrome-Induced Accumulation of cab RNA and rbcS RNA.

For several species of plants the abundance of those transcripts encoding the chlorophyll a/b binding protein (cab RNA) and the small subunit of ribulose-1,5-biphosphate carboxylase-oxygenase (rbcS RNA) has been established as being under the control of phytochrome. However, this conclusion does not take into account the various types of phytochrome control based on both the fluence of red light necessary to induce the response and the ability of far red light either to induce or to reverse the response. The fluence of red light necessary to induce the accumulation of rbcS RNA was found to be 10,000 times greater than that necessary to induce the accumulation of cab RNA. Furthermore, far red light alone was capable of inducing the accumulation of cab RNA. It is possible, therefore, that developing pea buds accumulate cab RNA before rbcS and that cab RNA is not subject to the normal end-of-day signals affecting many phytochrome responses.

Hurricane Allen's Impact on Jamaican Coral Reefs.

Coral reefs of north Jamaica, normally sheltered, were severely damaged by Hurricane Allen, the strongest Caribbean hurricane of this century. Immediate studies were made at Discovery Bay, where reef populations were already known in some detail. Data are presented to show how damage varied with the position and orientation of the substraturn and with the shape, size, and mechanical properties of exposed organisms. Data collected over succeeding weeks showed striking differences in the ability of organisms to heal and survive.

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.

Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum.

Prominent features of the cytosine methylation pattern of the Pisum sativum nuclear ribosomal RNA genes have been defined. Cytosine methylation within the C-C-G-G sequence was studied using the restriction enzymes HpaII and MspI and gel blot hybridizations of the restriction digests. The extent to which particular features of the methylation pattern change during seedling development has also been determined. Total cellular DNA, purified from defined sections of pea seedlings grown under different lighting conditions, was analyzed with DNA hybridization probes derived from different portions of a cloned member of the nuclear rRNA gene family. By use of an indirect end-labeling technique, a map of 23 cleavable HpaII and/or MspI sites in genomic rDNA was constructed. The map covers about 90% of the rDNA repeat including the entire non-transcribed spacer region and most of the rRNA coding sequences. One notable feature of the map is that the most prominent HpaII site, located about 800 base-pairs upstream from the 5' end of the mature 18 S rRNA, is cleaved only in one of the two most abundant rDNA length variants (the short variant). With a gel blot assay specific for cleavage at this site, we estimated the HpaII sensitivity of DNA preparations from several stages of pea seedling development. We find that, while methylation is generally low in young seedlings, DNA obtained from the apical buds of pea seedlings is highly methylated. Further, the methylation level of rDNA within the pea bud decreases as the buds are allowed to develop under continuous white light. Our data, taken together with published studies on pea seedling development, indicate that cytosine methylation levels may be related to the regulated expression of the nuclear rRNA genes in pea.

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.

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.

PGO waves in rats in the non-paradoxical sleep states.

Experiments were conducted in rats in order to characterize further the occurrence of PGO waves recorded from the region of the locus coeruleus, which were elicited by auditory stimuli. Repeated stimulus presentations at regular interstimulus intervals resulted in a consistent, state-dependent pattern in the occurrence and amplitudes of elicited PGO waves. The probability of eliciting a PGO wave was lowest during paradoxical sleep and highest immediately afterwards. This was followed by a gradual decline in responding during the ensuing period of slow-wave sleep. These results were obtained regardless of at what point in the rats' sleep cycles presentations of the auditory stimuli began. In other experiments, a comparison of elicited PGO waves with the acoustic startle reflex revealed major differences between these 2 responses. PGO waves could be elicited with very low intensity stimuli that were below the threshold for producing a behavioral response, while much louder stimuli were required to elicit a startle. The startle reflex habituated in a linear fashion and did not display state-related changes. It was concluded that elicited PGO waves are an electrophysiological sign of sensory responsiveness, and that their spontaneous appearance during and preceding paradoxical sleep reflects endogenously generated reticular activation.

Spontaneous and elicited PGO spikes in rats.

High-amplitude waves, similar in distribution and configuration to ponto-geniculo-occipital (PGO) spikes recorded in cats, were recorded from the area of the locus coeruleus of chronically implanted, unanesthetized albino rats. The mean frequency of spiking during paradoxical sleep (PS) was 19 per min and the amplitudes of the spikes ranged from 0.07 to 0.28 mV. Based on these findings and those of a previous report, it was concluded that these spikes are homologous with PGO spikes recorded in cats. It was also found that spikes identical to the spontaneously occurring spikes of PS could be produced during wakefulness and in all states of sleep with external auditory stimulation. These elicited spikes were not accompanied by eye movements. Initial stimulus presentations resulted in EEG desynchronization, orienting movements and PGO spikes. With continued stimulus presentations, EEG and other signs of alerting would habituate followed by a gradual habituation of the spikes. The latency to the appearance of an evoked spike ranged from 20 to 24 msec. These findings are in agreement with recent studies in cats showing that PGO spikes occur in response to external, auditory stimuli in wakefulness and all stages of sleep in addition to their spontaneous occurrence just prior to and during PS.

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.

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.

Parachlorophenylalanine does not affect pontine-geniculate-occipital waves in rats despite significant effects on other sleep-waking parameters.

The effects of 376 mg/kg parachlorophenylalanine (PCPA) methyl ester on spontaneous and elicited pontine-geniculate-occipital (PGO) waves, locomotor activity, and sleep-staging were studied in albino rats. It was found that PCPA had no effect on the frequency per minute of spontaneously occurring PGO waves or on the relationship between elicited PGO waves and the sleep-waking cycle, which has been reported in normal rats. In contrast, significant decreases in sleep, increases in spontaneous locomotor activity, and alterations in other spontaneously emitted behaviors were observed. It was concluded that PCPA does not affect PGO waves in rats in contrast to the dramatic effects of this drug on PGO waves in cats.

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.

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 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.

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.

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.

Phytochrome control of specific mRNA levels in developing pea buds : the presence of both very low fluence and low fluence responses.

We have examined phytochrome regulated changes in transcript abundance for 11 different light regulated mRNAs in developing pea buds. Fluence-response curves were measured for changes in transcript abundance in response to red light pulses in both the low and very low fluence ranges. Most transcripts show only low fluence responses, with a threshold of approximately 10 micromoles per square meter. All of the low fluence responses are reversible by far red light. One transcript shows a very low fluence response, with a threshold of approximately 10(-4) micromoles per square meter. As expected, the very low fluence response is not far red reversible and in fact can be induced by far red light.Various fluences of red light were also used as pretreatments before transferring seedlings to continuous white light. One transcript responds to pretreatments in the very low fluence range, several respond to pretreatments in the low fluence range (including chlorophyll a/b binding protein RNA and ribulose-1,5-bisphosphate carboxylase RNA), and several show no response to the red light under these conditions. The threshold of these low fluence responses is approximately 10(2) micromoles per square meter, one order of magnitude greater than the threshold of the low fluence responses to red light alone.The transcripts may also be grouped by their responses to white light treatment alone. Three of the clones correspond to transcripts whose abundance decreases after a 24 hour white light treatment. The remainder of the mRNAs increase between 2- and 10-fold in response to the 24 hour white light.