Blue Light-responsive Promoter Research Articles

<i>Trans</i>-Factor PTF1 Participates in the Response to Salinity but Does Not Regulate Expression of the <i>psbD</i> Gene in <i>Arabidopsis thaliana</i>

The existing data on the role of PTF1/TCP13 belonging to the TCP family of transcription factors in regulating expression of a psbD plastid gene encoding a D2 protein of PSII are controversial. To analyze biological functions of PTF1/TCP13, transformed plants expressing PTF1/TCP13 under a -estradiolinducible promoter were used. PTF1/TCP13 overexpression did not provide the expected increase in the accumulation of psbD transcripts transcribed from BLRP (Blue Light Responsive Promoter), though their level significantly increased under exposure to light or abscisic acid (ABA). PTF1/TCP13 was up-regulated by ABA; moreover, genes of the canonic pathway of the ABA signal transduction were involved in the regulation of PTF1/TCP13 expression. In addition, PTF1/TCP13 was induced in response to salt stress However, in the overexpressing line, salt tolerance and expression of salt stress markers, as well as a number of genes for the synthesis and signaling of ABA, were reduced compared to plants with the normal level of expression of this transcription factor, that is, PTF1/TCP13 acted as a negative regulator of salt stress Thus, PTF1 does not belong to plastid transcription factors. Nevertheless, it represents one of the components of the ABA-dependent regulatory chain capable of modifying expression of nuclear and chloroplast genes in response to changes in homeostasis.

Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription.

The UV-B photoreceptor UVR8 promotes photosynthetic efficiency in Arabidopsis thaliana exposed to elevated levels of UV-B

The UV-B photoreceptor UVR8 regulates expression of genes in response to UV-B, some encoding chloroplast proteins, but the importance of UVR8 in maintaining photosynthetic competence is unknown. The maximum quantum yield of PSII (F (v)/F(m)) and the operating efficiency of PSII (Φ(PSII)) were measured in wild-type and uvr8 mutant Arabidopsis thaliana. The importance of specific UVR8-regulated genes in maintaining photosynthetic competence was examined using mutants. Both F (v)/F(m) and Φ(PSII) decreased when plants were exposed to elevated UV-B, in general more so in uvr8 mutant plants than wild-type. UV-B increased the level of psbD-BLRP (blue light responsive promoter) transcripts, encoding the PSII D2 protein. This increase was mediated by the UVR8-regulated chloroplast RNA polymerase sigma factor SIG5, but SIG5 was not required to maintain photosynthetic efficiency at elevated UV-B. Levels of the D1 protein of PSII decreased markedly when plants were exposed to elevated UV-B, but there was no significant difference between wild-type and uvr8 under conditions where the mutant showed increased photoinhibition. The results show that UVR8 promotes photosynthetic efficiency at elevated levels of UV-B. Loss of the DI polypeptide is probably important in causing photoinhibition, but does not entirely explain the reduced photosynthetic efficiency of the uvr8 mutant compared to wild-type.

Light induction of Arabidopsis SIG1 and SIG5 transcripts in mature leaves: differential roles of cryptochrome 1 and cryptochrome 2 and dual function of SIG5 in the recognition of plastid promoters

In higher plants, multiple nuclear-encoded sigma factors activate select subsets of plastid gene promoters in a partially redundant manner. We analysed the light induction profiles of transcripts from six Arabidopsis sigma factor (AtSIG) genes in mature leaves, focusing on the effects of wavelength and intensity. Red-light illumination (660 nm) of dark-adapted plants strongly induced AtSIG1 transcripts, while blue-light illumination (470 nm) caused strong and rapid induction of AtSIG1 and AtSIG5 transcripts. The fluence response differed in blue-light-responsive rapid induction in AtSIG1 and AtSIG5. AtSIG1 transcripts increased to plateau with a threshold of 2 micromol m(-2) sec(-1) under all fluences examined (1-50 micromol m(-2) sec(-1)), and AtSIG5 transcripts were induced with a distinct two-phase profile, with the lower-fluence induction similar to that of AtSIG1 and further enhancement with increasing fluences greater than 10 micromol m(-2) sec(-1). Blue-light-receptor mutational analysis revealed that AtSIG5-specific two-phase induction is mediated through cryptochrome 1 and cryptochrome 2 at lower fluences and more significantly through cryptochrome 1 at higher fluences. In mature chloroplasts, the promoters of psbA and psbD are predominantly recognized by AtSIG5 among six sigma factors. Using a protoplast transient expression assay with AtSIG5-AtSIG1 chimeric genes, we present evidence that AtSIG5 contains determinants for activating the psbD blue-light-responsive promoter (BLRP) in region 4.2 rather than region 2.4. Amino acid scanning within AtSIG5 region 4.2 revealed that Asn484, but not Arg493, functions as a key residue for psbD BLRP activation. Arginine 493 may be involved in psbA promoter recognition.

Plastid Transcription in Higher Plants

The plastid genome is transcribed by nucleus-encoded (NEP) and plastid encoded (PEP) RNA polymerases. NEP transcribes housekeeping genes as well as genes coding for PEP core subunits and its activity is replaced by PEP in chloroplasts resulting in differential expression of genes in a developmental context. PEP is a prokaryotic-type enzyme in which nuclear-encoded σ factors function as promoter recognition subunit. A phylogenetic analysis for σ factors identified so far in plants shows that plant σ factors are members of bacterial σ70 family and divided into six groups, Sig1 through Sig6, which are integrated into four clusters consisting of Sig1 and Sig4, Sig2 and Sig3, Sig5 and Sig6. All plastid σ factors recognize bacterial σ70-type promoters, but they differ in promoter preference and the tissue-, developmental stage- and environmental-dependent expression. Sig5 is distinct from the other σ factors in its structure, function, and expression in response to light and stress. A promoter of the psbD operon, psbD blue light responsive promoter (psbDBLRP) is a typical example that is under the control of a combination of various signals arising in the nucleus and plastids in response to the tissue specific and developmental stage- and environment-dependent cues. psbDBLRP is recognized only by Sig5, which is expressed by a cryptochrome-mediated blue light signal and signals responding to stress conditions. The activity of psbDBLRP is also under the control of circadian clock. Furthermore, it may be regulated by redox signals generated by photosynthetic electron transport in the chloroplast presumably through the change of the binding affinity of a nuclear encoded transcription factor for the enhancer element located upstream of the core promoter region of the psbD operon.

Two independent light signals cooperate in the activation of the plastid psbD blue light-responsive promoter in Arabidopsis

The psbD blue light-responsive promoter (BLRP), whose activation has been considered to require strong blue light, is recognized only by SIG5 among six σ factors of plastid RNA polymerase in Arabidopsis. We found SIG5 transcript accumulation was rapidly induced after a 30-min induction time by blue light (470 nm) with an intensity threshold of 5 μmol m −2 s −1 through cryptochromes. Besides this weak blue light, the psbD BLRP activation required the stronger light such as 50 μmol m −2 s −1 irrespective of blue or red light (660 nm). Thus, the two independent light signalings, the cryptochrome-mediated signaling to induce SIG5 transcription and the stronger light-dependent signaling, cooperate to activate the psbD BLRP.

The multiple-stress responsive plastid sigma factor, SIG5, directs activation of the psbD blue light-responsive promoter (BLRP) in Arabidopsis thaliana.

Transcription in higher plant plastids is performed by two types of RNA polymerases called NEP and PEP, and expression of photosynthesis genes in chloroplasts is largely dependent on PEP, a eubacteria-type multi-subunit enzyme. The transcription specificity of PEP is modulated by six nuclear-encoded sigma factors (SIG1 to SIG6) in Arabidopsis thaliana. Here, we show that one of the six sigma factors, SIG5, is induced under various stress conditions, such as high light, low temperature, high salt and high osmotic conditions. Interestingly, transcription from the psbD blue light-responsive promoter (psbD-BLRP) was activated by not only light but also various stresses, and the transcription and the transcriptional activation of psbD-BLRP were abolished in a sig5-2 mutant. This suggests that the PEP holoenzyme containing SIG5 transcribes the psbD-BLRP in response to multiple stresses. Since the seed germination under saline conditions and recovery from damage to the PSII induced by high light were delayed in the sig5-2 mutant, we postulate that SIG5 protects plants from stresses by enhancing repair of the PSII reaction center.

Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor, AtSig5.

Light is one of the most important environmental factors regulating expression of photosynthesis genes. The plastid psbD gene encoding the photosystem II reaction center protein D2 is under the control of a unique blue light responsive promoter (BLRP) that is transcribed by a bacterial-type plastid RNA polymerase (PEP). Promoter recognition of PEP is mediated by one of the six nuclear-encoded sigma factors in Arabidopsis. The replacement of the plastid sigma factor associated with PEP may be the major mechanism for switching of plastid transcription pattern in response to environmental and developmental signals. This study demonstrates that AtSig5 is a unique sigma factor that is essential for psbD BLRP activity. A T-DNA insertional mutant with reduced AtSIG5 expression resulted in loss of primary transcripts from the psbD BLRP. Furthermore, transient overexpression of AtSig5 in dark-adapted protoplasts specifically elevated psbD and psbA transcription activities. On the other hand, overproduction of AtSig2 enhanced the transcription of psbA gene and trnE operon, but not psbD transcription. The AtSIG5 gene is phylogenetically distinct from other plastid sigma factors, and its expression is induced exclusively by blue light. We propose that AtSig5 acts as a mediator of blue light signaling that specifically activates the psbD BLRP in response to blue light in Arabidopsis.

Expression of a higher-plant chloroplast psbD promoter in a cyanobacterium (Synechococcus sp. strain PCC7942) reveals a conserved cis-element, designated PGT, that differentially interacts with sequence-specific binding factors during leaf development.

The chloroplast psbD gene, which encodes the D2 subunit of photosystem II, is regulated by a blue light-responsive promoter (BLRP). We tested the ability of different regions of the barley (Hordeum vulagare) BLRP to drive transcription of the lacZ reporter gene in genomic transformants of Synechococcus sp. strain PCC7942. The barley BLRP was transcribed in Synechococcus from the same initiation sites that are used in plant chloroplasts in vivo. A region of the BLRP, residing between -83 and -112 bp upstream from the transcription initiation sites, functioned as a negative element in Synechococcus. Nucleotide sequences within this region are conserved among the psbD genes of several monocots and dicots, and with the nuclear negative regulatory element GT. Thus this new cis-element was designated Plastid GT, PGT. Proteins from chloroplasts of barley and Arabidopsis thaliana interacted with PGT in a sequence-specific and developmental-dependent manner. The DNA-protein complexes from Arabidopsis chloroplasts are composed of 60- and 38-kDa polypeptides. We postulate that GT and PGT have evolved in the nucleus and chloroplast, respectively, from a common ancestral regulatory element.

Detailed Architecture of the Barley ChloroplastpsbD-psbC Blue Light-responsive Promoter

The photosystem II reaction center chlorophyll protein D2, is encoded by the chloroplast gene psbD. PsbD is transcribed from at least three different promoters, one which is activated by high fluence blue light. Sequences within 130 base pairs (bp) of the psbD blue light-responsive promoter (BLRP) are highly conserved in higher plants. In this study, the structure of the psbD BLRP was analyzed in detail using deletion and site-directed mutagenesis and in vitro transcription. Deletion analysis showed that a 53-bp DNA region of the psbD BLRP, from -57 to -5, was sufficient for transcription in vitro. Mutation of a putative prokaryotic -10 element (TATTCT) located from -7 to -12 inhibited transcription from the psbD BLRP. In contrast, mutation of a putative prokaryotic -35 element, had no influence on transcription. Mutation of a TATATA sequence located between the barley psbA -10 and -35 elements significantly reduced transcription from this promoter. However, site-directed mutation of sequences located between -35 and -10 had no effect on transcription from the psbD BLRP. Transcription from the psbD BLRP was previously shown to require a 22-bp sequence, termed the AAG-box, located between -36 and -57. The AAG-box specifically binds the protein complex AGF. Site-directed mutagenesis identified two different sequence motifs in the AAG-box that are important for transcription in vitro. Based on these results, we propose that positive factors bind to the AAG-box and interact with the chloroplast-encoded RNA polymerase to promote transcription from the psbD BLRP. Transcription from the psbD BLRP is thus similar to type II bacterial promoters that use activating proteins to stimulate transcription. Transcription of the psbD BLRP was approximately 6. 5-fold greater in plastid extracts from illuminated versus dark-grown plants. This suggests that light-induced activation of this promoter in vivo involves factors interacting with the 53-bp psbD BLRP in vitro.

DET1 represses a chloroplast blue light-responsive promoter in a developmental and tissue-specific manner in Arabidopsis thaliana.

The chloroplast psbD-psbC loci, which encode the D2 and CP43 subunits of the photosystem II reaction center, respectively, are regulated by a blue light-responsive promoter (BLRP). It has recently been shown in barley seedlings that activation of psbD-psbC transcription by blue light involves inhibition of a protein kinase that represses the BLRP in the dark. To elucidate further the photosensory pathways regulating the psbD BLRP, the effects of three nuclear mutations on the expression of the BLRP in chloroplasts of Arabidopsis thaliana were examined. The mutants used included the det1-1 and det1-6 alleles for the nuclear protein DET1, involved in repressing photomorphogenesis, and the cry1 gene for the blue light photoreceptor, cryptochrome (CRY1), involved in hypocotyl elongation. The BLRP was not significantly expressed in cotyledons of light-grown wild-type seedlings, unlike the light-responsive expression of the chloroplast, psbA and rbcL, and nuclear, Lhcb and Chs, genes. Analysis of the mutants revealed that DET1 represses transcription from the BLRP in a developmental and tissue-specific manner, which is unique from the effects that DET1 has on other light-regulated promoters. In addition, the cry1 mutation did not reduce the expression of the BLRP in response to blue light. This suggests that the BLRP is regulated by a different photosensory system relative to CRY1. A model is proposed involving blue light, DET1 and phytochrome in regulating transcription from the psbD BLRP.

Structure and blue-light-responsive transcription of a chloroplast psbD promoter from Arabidopsis thaliana.

We characterized the effects of light on psbD transcription and mRNA levels during chloroplast development in Arabidopsis thaliana. After 6 to 12 hours of illumination of dark-grown seedlings, two psbD mRNAs were detected and their 5' ends were mapped to positions -550 and -190 bp upstream from the psbD translational start codon. Their kinetics of accumulation resembled the accumulation of chloroplast psbA and rbcL mRNAs but differed from the accumulation of the nuclear-encoded Lhcb and Chs mRNAs. A third psbD mRNA with its 5' ends at position -950 accumulated after illumination of > 180 h. The 5' ends of this transcript were mapped to a nucleotide sequence that is highly conserved with functional sequences in the barley (Hordeum vulgare) blue-light-responsive promoter (BLRP). Transcription from the Arabidopsis psbD promoter was 3-fold higher in blue relative to red light, whereas red and blue light affected total chloroplast, rbcL, and 16S rDNA transcription similarly. This study shows that transcription of Arabidopsis psbD is mediated by a BLRP and suggests that psbD genes in other land plants are regulated by a common blue-light-signaling pathway. Isolating the BLRP from Arabidopsis will allow molecular genetic studies aimed at identifying the pertinent photoreceptor and components of this phototransduction pathway.

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.

Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

The plastid gene psbD encodes the photosystem II reaction center chlorophyll protein D2. psbD is located in a complex operon that includes psbC, psbK, psbl, orf62, and trnG. The operon is transcribed from at least three different promoters. One of the psbD promoters is differentially activated when plants are exposed to blue light. In this study, the psbD blue light-responsive promoter was accurately transcribed in vitro in high-salt extracts of barley plastids. Transcription required supercoiled templates and was inhibited by tagetitoxin, an inhibitor of plastid transcription. Escherichia coli RNA polymerase did not recognize the psbD light-responsive promoter with the same specificity as plastid RNA polymerase. Deletion analyses demonstrated that sequences between -39 and -68, upstream of the transcription initiation site, were required for transcription of the psbD blue light-responsive promoter. This DNA region is highly conserved among plant species and contains multiple AAG sequences. Gel shift assays and DNase I footprinting experiments demonstrated that the AAG-rich DNA sequence interacts with a sequence-specific DNA binding factor termed AGF. Point mutations in the AAG cis element decreased binding of AGF and inhibited transcription from the psbD light-responsive promoter. We concluded that AGF is an essential factor required for transcription of the psbD light-responsive promoter.

A novel light-regulated promoter is conserved in cereal and dicot chloroplasts.

The chloroplast psbD-psbC genes encode D2 and cp43, a reaction center protein and chlorophyll-binding antenna protein of photosystem II, respectively. We have previously shown that differential accumulation of light-induced psbD-psbC mRNAs in barley chloroplasts is due to transcription from a blue light-responsive promoter (LRP). It is hypothesized that the light-induced mRNAs help to maintain levels of the D2 polypeptide, which is photodamaged and degraded in illuminated plants. To determine if light-induced accumulation of psbD-psbC mRNAs was a conserved phenomenon in chloroplasts, the expression of psbD-psbC operons from five cereals (barley, wheat, rice, maize, and sorghum) and three dicot (tobacco, spinach, and pea) species was examined. Cereal and dicot psbD-psbC operons differ due to several DNA rearrangements that moved psbK-psbI proximal to psbD-psbC, allowing cotranscription of these genes and production of several unique transcripts in cereals. Despite differences in the structure and expression of the cereal and dicot psbD-psbC operons, the accumulation of light-induced psbD-psbC mRNAs was conserved in all species studied. An unusual feature of the light-induced mRNAs was the occurrence of 5' end microheterogeneity. The multiple 5' termini were mapped to several consecutive nucleotides (8 to 25 bp) within a highly conserved (61%) DNA region that represents the transcription initiation site for the mRNAs in barley and tobacco. The novel LRP differs in sequence from typical plastid promoters that have prokaryotic "-10" and "-35" elements and is centered 570 bp (cereals), 900 bp (tobacco, spinach), or 1100 bp (pea) upstream from the psbD translational start codon. We propose that physiological and gene regulatory demands of the chloroplast act as constraints that preserved the linkage of the LRP with psbD despite DNA inversions involving the psbD upstream region.