Lhcb Genes Research Articles

Low temperature regulates Arabidopsis Lhcb gene expression in a light-independent manner.

Low temperature treatment of dark-grown seedlings of Arabidopsis thaliana results in a rapid increase in the amount of mRNAs encoding for the major polypeptides of the light-harvesting complex of photosystem II (Lhcb1 genes). This increase is transient and seems to be due mainly to the accumulation of Lhcb1*3 transcripts, indicating that low temperature differentially regulates the expression of the Arabidopsis Lhcb1 gene family in the dark. A 1.34 kb fragment of the Lhcb1*3 promoter is sufficient to confer low temperature regulation to a reporter gene in transgenic Arabidopsis etiolated seedlings, suggesting that the regulation is occurring at the transcriptional level. The cold-induced accumulation of Lhcb1*3 mRNA is not part of a general response to stressful conditions since no accumulation is detected in response to water stress, anaerobiosis or salt stress. The amount of Lhcb1*3 mRNA decrease in response to exogenous abscisic acid (ABA) suggesting that this phytohormone acts as a negative regulator. Moreover, the accumulation of Lhcb1*3 mRNAs in cold-treated ABA deficient etiolated seedlings is higher than that of wild-type and ABA insensitive etiolated seedlings, indicating that low temperature regulation of Lhcb1*3 is not mediated by ABA.

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

We have isolated the gene for a protein designated CCA1. This protein can bind to a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb1*3, which is necessary for its regulation by phytochrome. The CCA1 protein interacted with two imperfect repeats in the Lhcb1*3 promoter, AAA/cAATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology to the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding to the Lhcb1*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCA1 showed reduced phytochrome induction of the endogenous Lhcb1*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-1A, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was not affected. Thus, the CCA1 protein acts as a specific activator of Lhcb1*3 transcription in response to brief red illumination. The expression of CCA1 RNA was itself transiently increased when etiolated seedlings were transferred to light. We conclude that the CCA1 protein is a key element in the functioning of the phytochrome signal transduction pathway leading to increased transcription of this Lhcb gene in Arabidopsis.

Involvement of protein kinase and extraplastidic serine/threonine protein phosphatases in signaling pathways regulating plastid transcription and the psbD blue light-responsive promoter in barley.

We investigated the signaling pathways that control changes in plastid transcription in response to development and light. Plastid gene expression was analyzed in dark-grown barley (Hordeum vulgare L.) seedlings treated in vivo with an inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), or an inhibitor of protein kinases (K252a), followed by exposure of the seedlings to either red, blue, or white light. OA prevented blue light from activating the plastid pshD blue-light-responsive promoter (BLRP) and prevented red and blue light from activating the expression of the plastid-encoded rbcl and psbA and the nuclear-encoded RbcS and Lhcb genes. OA reduced total plastid transcription activity in dark- and light-grown seedlings by 77 to 80%, indicating that OA prevented light-responsive transcription by reducing total plastid transcription. In contrast, K252a activated the accumulation of mRNAs arising from the BLRP. Blue light in combination with K252a increased psbD mRNA levels in an additive manner. The results indicate that protein phosphatases 1 and/or 2A, which reside external to the organelle, are required for proper function of plastid transcription and chloroplast development, whereas a protein kinase represses the BLRP in plants grown in the dark.

A Myb-Related Transcription Factor Is Involved in the Phytochrome Regulation of an Arabidopsis Lhcb Gene

A Myb-Related Transcription Factor Is Involved in the Phytochrome Regulation of an Arabidopsis Lhcb Gene

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.

Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants.

A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni(2+)-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-A resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system. Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.

Lhcb Transcription Is Coordinated with Cell Size and Chlorophyll Accumulation (Studies on Fluorescence-Activated, Cell-Sorter-Purified Single Cells from Wild-Type and immutans Arabidopsis thaliana).

To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined [beta]-glucuronidase (GUS) enzyme activities, chlorophyll contents, and cell sizes in fluorescence-activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the green sectors to compensate for a lack of photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis.

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.

A DNA binding activity for one of two closely defined phytochrome regulatory elements in an Lhcb promoter is more abundant in etiolated than in green plants.

The Lhcb2*1 gene of Lemna gibba is regulated positively by phytochrome, and two separate, 10-bp regions of this promoter have been shown to be necessary for phytochrome regulation. We have now analyzed the effects of one and two base pair mutations to define exactly two cis elements within these regions that are necessary for phytochrome regulation. These elements, designated REalpha and REbeta, consist in part of sequences highly conserved among promoter of genes encoding light-harvesting chlorophyll a/b proteins of photosystem II (Lhcb genes). They are located -134 to -129 bp and -114 to -109 bp from the transcription start site, respectively. REalpha has the sequence AACCAA and was found to interact specifically in vitro with a DNA binding activity in whole-cell extracts of plants. This activity was high in etiolated plants but much lower in green plants. REbeta has the sequence CGGATA. A GATA sequence created at a position six nucleotides upstream could replace the function of REbeta. We conclude that the phytochrome regulation of Lhcb2*1 is mediated by at least two cis elements. These elements are likely to function by repression of the promoter activity in darkness, although the REbeta region also may be able to play a role in the activation of transcription.

Alterations in chlorophyll a/b binding proteins in Solanaceae cybrids.

In this study we have constructed a number of plants (cybrids), in which the nuclear genome of Nicotiana plumbaginifolia is combined with the plastome of Atropa belladonna, or the nuclear genome of N. tabacum with plastomes of Lycium barbarum, Scopolia carniolica, Physochlaine officinalis or Nolana paradoxa. Our biochemical and immunological analyses prove that in these cybrids the biogenesis of the chlorophyll a/b binding proteins (CAB) of the light harvesting complex II (LHCII) is altered. Besides normal sized CAB polypeptides of 27, 25.5 and 25 kDa, which become less abundant, the cybrids analyzed have additional polypeptides of 26, 24.5 and 24 kDa. Direct protein micro-sequencing showed that at least two truncated 26 kDa CAB polypeptides in plant cells containing a nucleus of N. plumbaginifolia and plastids of A. belladonna are encoded by the type 1 Lhcb genes. These polypeptides are 11-12 amino acids shorter at the N-terminus than the expected size. Based on the available data we conclude that the biogenesis of the LHCII in vivo may depend on plastome-encoded factor(s). These results suggest that plastome-encoded factors that cause specific protein degradation and/or abnormal processing might determine compartmental genetic incompatibility in plants.

The greening process in cress seedlings IV. Light regulated expression of single Lhc genes

Transcript levels of single Lhc genes were determined in etiolated cress seedlings ( Lepidium sativum L.) after various irradiations. Hybridization probes used in this study specifically detected transcripts of three Lhcb1 genes (probes cd1, cd2, cd3), one Lhcb2 gene (probe cd4), one Lhcb3 gene (probe cd5) and one Lhcb3 gene (probe 6). A 2 min pulse of red light resulted in a transient increase in the steady-state level of all transcripts determined for up to 10 h after the light pulse. Light induction was stronger in 4 day old than in 3 day old seedlings. The maximum of light induction was generally reached 4 h after a pulse of red light except for Lchb2 and Lchb3 transcripts that exhibited a maximum already 2 h after the light pulse. The relationship between red, blue and far-red irradiation was similar for all investigated Lhc genes: transcrip levels were 75–85% after a 30 min blue and 60–70% after a 30 min far-red pulse, compared with the red pulse = 100%. The contribution of the photoreceptor phytochrome and the physiological role of different kinetics of expression are discussed.

Chloroplast Response in Dunaliella salina to Irradiance Stress (Effect on Thylakoid Membrane Protein Assembly and Function).

The chloroplast response in the green alga Dunaliella salina to irradiance stress was investigated. Cells were grown under low light (LL) at 100 [mu]mol photons m-2 s-1 or high light (HL) at 2000 [mu]mol photons m-2 s-1 incident intensity. LL-grown cells had a low chlorophyll (Chl) a/b ratio, an abundance of light-harvesting complex II proteins (LHC-II), and a large Chl antenna size. HL-grown cells had a higher Chl a/b ratio, relatively fewer LHC-II, and a small Chl antenna size. The more abundant higher molecular mass subunits of the LHC-II (approximately 31 kD) were selectively depleted from the thylakoid membrane of HL-grown cells. Light-shift experiments defined the kinetics of change in the subunit composition of the LHC-II and suggested distinct mechanisms in the acclimation of thylakoids to HL or LL conditions. The results showed that irradiance exerts a differential regulation on the expression of various Lhcb genes. The specific polyclonal antibodies used in this work, raised against the purified LHC-II, cross-reacted with a polypeptide of approximately 20 kD in HL-grown samples. In this work we examined the dynamics of induction of this novel protein and discuss its function in terms of a chloroplast response to the level of irradiance.

A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway

One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nial*2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1*3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the transformed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1*2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.

Characterization of a Lhcb5 cDNA from Scots pine (Pinus sylvestris).

Despite extensive studies on the LHC proteins of PSI and PSII (for review, see Jansson, 1994), the detailed polypeptide composition of LHC I and LHC I1 has not been understood until recently. The nature of the Lhcb5 gene product has been one of the major uncertainties, since it has been identified both as the CP26 (or the equivalent complex LHCIIc) polypeptide and one of two CP29 polypeptides (Jansson et al., 1992; Morishige and Thomber, 1992). Lhcb5 genes have previously been characterized from tomato (Pichersky et al., 1991) and barley (Sorensen et al., 1992); here we present the characterization of a Lhcb5 cDNA clone from the gymnosperm Scots pine (Pinus sylvestris) (Table I). Using a tomato Lhcb5 cDNA (Pichersky et al., 1991) as probe, we isolated partia1 Lhcb5 cDNAs, which subsequently were used to find a full-length cDNA, encoding a precursor Lhcb5 protein of 302 amino aads. The transit peptide cleavage site is not known for this protein due to N-terminal blockage of the mature polypeptide (Pichersky et al., 1991). A tentative cleavage site after Se?', which would give rise to a mature protein with a molecular mass of 27.1 kD, has recently been suggested (Jansson, 1994). Southem blotting showed that Lhcb5 is a single-copy gene in Scots pine, named Lhcb5.1, according to Jansson et al. (1992). We also investigated the light regulation of the Scots pine Lhca and Lhcb genes by transferring dark-grown Scots pine seedlings to light and measuring the N subsequent light treatment did not result in further induction. We found a general correspondence between Chl synthesis and expression of the Lhca and Lhcb genes, similar to what Canovas et al. (1993) reported for Pinus palustris seedlings. In conifers Chl synthesis, rather than light per se, seems to be a major determinant for LhcalLhcb gene regulation.

On the Expression of Several Lhc Genes in Garden Cress (Lepidium sativum L.)

The polymerase chain reaction was used to prepare gene-specific probes for several Lhc genes coding for chlorophyll a/b-binding proteins of cress (Lepidium sativum L.). Due to the presence of about 150 basepairs of the coding region, the isolated clones could be attributed to Lhc a3 (1 clone), Lhc b1 (5 clones), Lhc b2 (1 clone) and Lhc b3 (1 clone) genes. Probes prepared from the 3'non-coding regions of the clones did not cross-hybridize; they were specific for 3 different Lhc b1 transcripts and one each of Lhc b2, Lhc b3 and Lhc a3 transcripts. The transcript levels were higher in leaves than in cotyledons of light-grown seedlings; they decreased significantly in cotyledons from week 1 to week 4. The levels of 2 Lhc b1 transcripts (detected with probes cd1 and cd2) changed from 1 week old cotyledons (30% cd1, 28% cd2) to 3 months old leaves (14% cd1), 44% cd2), stems (11% cd1, 56% cd2) and fruits (15% cd1, 62% cd2, all values percent of total transcripts), whereas transcript levels of another Lhc b1 gene (detected with probe cd3) and of a Lhc a3 gene remained nearly constant. The level of Lhc b2 and Lhc b3 transcripts were 1-2 orders of magnitude smaller than those of the other Lhc transcripts. The data obtained with cress plants are compared with published data from other plants.

Two 10-bp regions are critical for phytochrome regulation of a Lemna gibba Lhcb gene promoter.

Two small regions of the promoter of an Lhcb gene encoding a light-harvesting chlorophyll a/b protein were identified as essential in conferring phytochrome responsiveness by using a transient expression assay. Initially, 5' deletion analysis of cabAB19, an Lhcb2 gene of Lemna, showed that sequences within the region from -174 to -104 relative to the start of transcription were necessary for phytochrome regulation. Internal deletion and substitution mutants were used to demonstrate that no additional phytochrome-responsive regions exist between -1600 and -174 in this promoter. A 171-bp fragment of the promoter extending from -239 to -69 was sufficient to impart phytochrome responsiveness to a minimal ubiquitin promoter that was not itself regulated by light. Specific binding of Lemna proteins to the region necessary for phytochrome responsiveness was demonstrated using in vitro polyacrylamide gel mobility shift assays and 1,10-phenanthroline copper ion footprinting. Further analysis of the region from -174 to -104 demonstrated that mutations in two separate 10-bp sequences, from -134 to -125 and from -114 to -105, could abolish phytochrome responsiveness; thus, there are two unique regions that are necessary for phytochrome regulation of this gene. One of these regions contains a CCAAT motif and the other a GATA motif. These motifs are conserved in the promoters of many Lhcb genes and may be important elements in the phytochrome responsiveness of this gene family.

Two 10-bp Regions Are Critical for Phytochrome Regulation of a Lemna gibba Lhcb Gene Promoter

Two 10-bp Regions Are Critical for Phytochrome Regulation of a Lemna gibba Lhcb Gene Promoter