Origins of phytochrome-modulated Lhcb mRNA expression in seed plants.

The levels of Lhcb mRNA in higher plants are regulated by phytochrome, cryptochrome, and an endogenous circadian oscillator. To determine whether similar regulatory mechanisms operate in the ancient gymnosperm Ginkgo biloba, we measured Lhcb mRNA levels in seedlings in response to different light conditions. Removal of a diurnally oscillating light stimulus caused dampening of maximal Lhcb mRNA accumulation levels, with little change in periodicity. Although low fluence pulses of both red and blue light given to etiolated seedlings caused maximal accumulation of Lhcb mRNAs characteristic of the phasic/circadian response seen in flowering plants, the additional initial acute response seen in flowering plants was absent. The induction of Lhcb gene expression in both cases was at least partially reversible by far-red light, and appeared biphasic over a range of red fluences. Together, these data indicate that Lhcb genes in G. biloba appear to be regulated in a manner similar to that of flowering plants, whereas signaling and attenuation of mRNA levels through the photoreceptor systems and circadian clock show features distinct from those characterized to date. The implications for these findings are discussed in light of the evolution of circadian clock input signaling.

Spatial patterns of phytochrome expression in young leaves of the fern Adiantum capillus-veneris.

The spatial distribution of phytochrome (PHY1) mRNA in the fern Adiantum was investigated by in situ hybridization. PHY1 mRNAs are expressed predominantly in the abaxial rather than the adaxial part of the petiole of leaf croziers. Moreover, the signals in light-grown croziers are predominantly nuclear in location rather than cytoplasmic.

Developmental and light-regulated expression of individual members of the light-harvesting complex b gene family in Pinus palustris.

Angiosperms requires light for multiple aspects of chloroplast development, including chlorophyll synthesis and induction of expression of the mRNAs encoding the major polypeptides of the light-harvesting complex of photosystem II (Lhcb genes). In contrast, many conifers, including pines, firs, and spruces, can accumulate chlorophyll and the light-harvesting chlorophyll a/b-binding proteins of photosystem II in complete darkness. To understand the factors responsible for the regulation of expression of individual Lhcb mRNAs in the pine Pinus palustris, we have prepared sequence-specific cDNA probes for each of three family members, Lhcb1*Pp1, Lhcb2*Pp1, and Lhcb2*Pp2, and have studied the expression of two of these, Lhcb1*Pp1 and Lhcb2*Pp2, in detail. The levels of expression of each sequence were disparate, and Lhcb1*Pp1-encoded transcripts were the most abundant in the light. Both Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs were expressed in stems and cotyledons, but Lhcb1*Pp1 mRNA was present at about 10-fold lower levels in stems than in cotyledons, in contrast to Lhcb2*Pp2 mRNA, which was expressed at higher levels in stems than in cotyledons. Both Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs were absent in embryos but were expressed during seedling development. The levels increased with age in both the light and the dark and in both cases were about 2-fold higher in the light than in the dark. Despite the expression of Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs during development in darkness, the levels of both mRNAs increased in dark-grown seedlings given red light in the low fluence range within 2 h of treatment.

Light-regulated and organ-specific expression of types 1, 2, and 3 light-harvesting complex b mRNAs in Ginkgo biloba.

In a prior study (E. Chinn and J. Silverthorne [1993] Plant Physiol 103: 727-732) we showed that the gymnosperm Ginkgo biloba was completely dependent on light for chlorophyll synthesis and chloroplast development and that expression of light-harvesting complex b (Lhcb) mRNAs was substantially increased by light. However, dark-grown seedlings that were transferred to constant white light took significantly longer than angiosperm seedlings to initiate a program of photomorphogenesis and the stems failed to green completely. We have prepared type-specific probes for mRNAs encoding major polypeptides of light-harvesting complex II (Lhcb1, Lhcb2, and Lhcb3) and have used these to analyze the expression of individual Lhcb mRNAs during greening. All three sequences accumulated in the top portions of dark-grown seedlings transferred to light, but, as was seen previously for total Lhcb mRNAs, there was a transient, reproducible decline in the levels of all three mRNAs after 4 d in the light. This transient decrease in Lhcb mRNA levels was not paralleled by a decrease in Chl accumulation. By contrast, there were significantly lower levels of all three Lhcb mRNAs in the lower portions of greening dark-grown stems as well as lower Chl levels. We conclude that although the tops of the plants have the capacity to etiolate and green, Gingko seedling stems continue a program of development into woody tissue in darkness that precludes greening when the seedlings are transferred to the light.

Organ-Specific Stability of Two Lemna rbcS mRNAs Is Determined Primarily in the Nuclear Compartment.

It has previously been shown that the organ-specific expression of two members of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene family is post-transcriptionally regulated in Lemna gibba. While both small subunit genes encoding SSU1 and SSU5B were transcribed at comparable levels in root and frond nuclei, SSU1 mRNA accumulated to high levels in both roots and fronds in contrast to SSU5B mRNA, which was of very low abundance in the roots compared with the fronds. In this study, we have used two approaches to pinpoint the step(s) at which SSU1 and SSU5B mRNAs are differentially accumulated in these organs. In the first approach, total nuclear steady state mRNA was isolated from roots and fronds, and the amount of each transcript was measured by RNase protection assays and compared with the transcription rates in isolated nuclei. In the second approach, cordycepin was used to inhibit mRNA synthesis in Lemna fronds or roots, and the rate of decay of each mRNA was measured by RNA gel blot analysis or RNase protection assays. Our findings indicate that the differential accumulation of SSU1 and SSU5B mRNAs in the fronds versus the roots is determined primarily in the nuclear compartment. In addition, SSU1 was found to have a longer half-life in total steady state mRNA than SSU5B had in both organs. This feature probably accounts for SSU1 being the predominantly expressed family member.

Light-dependent chloroplast development and expression of a light-harvesting chlorophyll a/b-binding protein gene in the gymnosperm Ginkgo biloba.

Unlike conifers, the gymnosperm Ginkgo biloba is dependent on light for chlorophyll (Chl) synthesis and initiation of chloroplast development. Dark-grown seedlings show complete etiolation, including no detectable Chl accumulation, no leaf expansion, and increased hypocotyl elongation. When dark-grown seedlings are placed in white light, Chl synthesis and leaf expansion are initiated, but unlike angiosperms, which initiate rapid photomorphogenesis, Ginkgo takes at least 1 week to change to a normal light-regulated pattern of growth. A cDNA clone (pLhcb*Gb1) encoding a Chl a/b-binding protein of light-harvesting complex II from Ginkgo mRNA has been used as a probe for the expression of this family of mRNAs. We have found that, in common with angiosperms but in marked contrast to pines, Lhcb mRNA is expressed in a highly light-dependent manner. In addition to being expressed in light-grown leaves, this sequence is also expressed in the green tissues of immature seeds. The Lhcb mRNA appears during greening in parallel with the onset of Chl synthesis. The complete sequence of pLhcb*Gb1 has been determined and the deduced amino acid sequence was found to be of type I based on comparison with signature sequences of angiosperm and gymnosperm sequences.

Light-independent synthesis of LHC IIb polypeptides and assembly of the major pigmented complexes during the initial stages of Pinus palustris seedling development.

Pinus palustris has a greatly reduced need for light to initiate chloroplast development in comparison to angiosperms. Light is not required for chlorophyll synthesis in dark-grown Pinus palustris seedlings. However, embryos do not contain chlorophyll, and synthesis is limited to seedlings having cotyledon lengths between about 0.5 cm and 2.0 cm. The final amount of chlorophyll accumulated by dark-grown seedlings is about one fifth of that in light-grown seedlingsat the same stage. The major light-harvesting chlorophyll a/b-polypeptides of Photosystem II (LHC IIb) are absent in the embryos but begin to accumulate in seedlings of 0.5 cm cotyledon length, irrespective of the light conditions. Although dark-grown seedlings accumulate most of the pigmented complexes seen in light-grown seedlings, there are differences in the subunit structure of some of them. These findings suggest that the majority of the components of the photosynthetic membrane do not require light for induction of synthesis or assembly into complexes, but that the final forms seen in light-grown seedlings may require light.

Post-transcriptional regulation of organ-specific expression of individual rbcS mRNAs in Lemna gibba.

Many studies of nuclear genes encoding chloroplast proteins have focused on the transcriptional regulation of their expression. The genes (rbcS) encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase, a major stromal protein, comprise one such group. We have examined the role played by post-transcriptional events in determining the relative levels of individual rbcS mRNAs in different organs of the aquatic monocot Lemna gibba. L. gibba is unusual among angiosperms in that its roots are normally exposed to light during growth and contain chloroplasts. We have found that such roots transcribe rbcS genes and contain rbcS mRNA. We have used sequence-specific probes from the 3'-untranslated region of six rbcS genes from L. gibba to analyze the expression of the individual genes in different organs. All six genes were expressed in steady-state mRNA in fronds grown in constant white light. However, only five of these were easily detectable in steady-state mRNA isolated from roots of the same plants, and the relative expression of each gene varied between the roots and the fronds. In steady-state mRNA, SSU1 was found to be highly expressed in both roots and fronds, whereas SSU40B was expressed at low levels in the roots as compared with the fronds, and SSU5B RNA was barely detectable in the roots. The extremely low level of SSU5B RNA in steady-state root mRNA is likely to be a consequence of post-transcriptional events because this gene was transcribed at comparable rates in vitro in nuclei isolated from either roots or fronds. Localization of individual gene transcripts by in situ hybridization showed that SSU1 and SSU5B are expressed in the same cells in the fronds. Thus, the mechanism of differential expression is likely to involve an organ-specific post-transcriptional mechanism.

Differential expression of individual genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in Lemna gibba.

The gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase in the monocot Lemna gibba contains approximately twelve members. We have isolated six of these genes from a genomic library, and sequenced five of the coding regions. The transit peptide nucleotide sequences are conserved, but less highly than the mature polypeptide coding sequence. The mature polypeptide amino acid sequences are identical to each other and to the sequence deduced from a cDNA clone derived from a seventh gene. Each of the five fully characterized genomic sequences contains a single intron in precisely the same position as the second intron of several dicots. The intron sequences differ in length and are less conserved than the coding sequences. The 3'-untranslated regions of the different genes have been sequenced and used to prepare gene-specific probes. These probes have been used to study the expression levels of individual rbcS sequences. Expression of six of the seven genes can be detected in total RNA isolated from plants grown in continuous light. The levels of RNA encoded by each expressed gene are regulated by the action of phytochrome, but there is variability in the amount of expression of each RNA.

Demonstration of transcriptional regulation of specific genes by phytochrome action.

We have developed an in vitro transcription system that uses nuclei isolated from Lemna gibba G-3. The in vitro transcripts include sequences homologous to hybridization probes for the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], the light-harvesting chlorophyll a/b-protein, and rRNA. Light-harvesting chlorophyll a/b-protein sequences are transcribed to a greater extent in nuclei isolated from plants grown in darkness with 2 min of red light every 8 hr than in nuclei isolated from dark-treated plants. Furthermore, the amount of these transcripts measured in plants given a single minute of red light after dark treatment is increased over the amount measured in dark-treated plants. The effect of red light is at least partially reversible by 10 min of far-red light given immediately after the red light pulse. Transcription of both rRNA and small subunit sequences is also stimulated by a single minute of red light as compared to dark-treated tissue. However, the relative magnitudes of the increases compared to the dark levels are smaller than the increase seen for the chlorophyll a/b-protein, possibly because of the higher level of transcription of these sequences in the dark. The effect of red light on the transcription of small subunit and rRNA sequences is also reversible by immediate treatment with 10 min of far-red light. Pulse chase studies of dark-treated nuclei for up to 110 min do not show substantial turnover of in vitro labeled small subunit and chlorophyll a/b-protein transcripts. We therefore conclude that phytochrome action has induced specific changes in transcription of these genes.

Immune complexes, gallium lung scans, and bronchoalveolar lavage in idiopathic interstitial pneumonitis-fibrosis.

We obtained results of lung immune complexes (LIC), circulating immune complexes (CIC), 48-hour gallium lung scans (scans), bronchoalveolar lavage (BAL), and pulmonary function tests in 20 patients with idiopathic interstitial pneumonitis-fibrosis. Sixteen patients had predominantly interstitial (13 cases UIP) and/or intraalveolar (3 cases DIP) cellular disease (group 1). Prior to corticosteroid therapy in group 1, scans were positive in 75 percent, CIC were elevated in 86 percent, LIC were present in 64 percent, and BAL was abnormal in 90 percent. Duration of follow-up after treatment was 3.5 +/- 1.0 year. In group 1 after treatment with corticosteroids in 13 patients and corticosteroids and penicillamine (three patients) and plasmapheresis (one patient), only four patients remain stable or improved. After corticosteroid therapy, elevated CIC returned to normal values despite progressive patient deterioration. In three patients, lung immune complexes were still detected after circulating immune complexes had returned to normal after corticosteroid therapy. In group 2 were four patients with fibrotic disease; scans and CIC were uniformly negative, LIC were weakly present in only one patient, and BAL was abnormal in all. Despite corticosteroid therapy, all have died or deteriorated. These results suggest that positive gallium lung scans, BAL, circulating immune complexes, and to a lesser extent, lung immune complexes are associated with the cellular phase of interstitial pneumonia, but do not reliably identify a corticosteroid-responsive group.

Phytochrome Control of the Expression of Two Nuclear Genes Encoding Chloroplast Proteins in Lemna gibba L. G-3.

Hybridization probes for two nuclear-coded chloroplast proteins of Lemna gibba L. G-3 have been constructed in order to investigate phytochrome regulation of specific sequences. The first probe is a cDNA clone encoding the small subunit of ribulose 1,5-bisphosphate carboxylase. This probe was isolated from a set of Lemna cDNA clones in the bacterial plasmid pBR322. The second probe is a subclone of a genomic clone encoding the light-harvesting chlorophyll a/b-protein. This clone was isolated from a set of genomic clones constructed in the lambda vector Charon 4 with L. gibba DNA fragments generated by partial EcoR1 digestion. The identity of these clones was confirmed by in vitro translation of RNA which hybridized to the cloned DNA. Plants grown under continuous white light contain high concentrations of both RNA sequences; however, when these plants are put into darkness the concentration of these RNAs decreases rapidly relative to the total amount of RNA. Plants grown in the dark with intermittent red light (2 minutes/8 hours) and put into complete darkness for 8 days also contain lower concentrations of the sequences in the total RNA. One minute of red light after this dark period results in a rapid increase in the levels of RNA hybridizing to the probes. The effect of red light can be reversed by far-red light. These experiments demonstrate that phytochrome action can rapidly influence either the rates of transcription or the rates of degradation of these mRNAs.

Protein synthesis in chloroplasts. VIII. Differential synthesis of chloroplast proteins during spinach leaf development.

Excised primary leaves of spinach (Spinacia oleracea) incorporate [35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.