Tetrapyrrole involvement in expression regulation of a nuclear gene of low-molecular-weight plastid protein ELIP

An inhibitor analysis was used for studying the tetrapyrrole role in the regulation of the expression of the nuclear gene encoding a low-molecular-weight protein, a stress plastid light-inducible protein ELIP. 2,2'-Dipyridyl and norflurazon were used as inhibitors. Experiments with dipyridyl demonstrated that tetrapyrroles were involved in the regulation of Elip gene expression, inhibiting it by approximately 50%. Similar results were obtained when there was photodestruction of the chloroplasts, caused by a plant treatment with norflurazon. The results confirm the involvement of the chloroplasts in the regulation of the nuclear gene expression coding for plastid proteins. Tetrapyrroles are important contributors to this process.     

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.     

'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.     

Tissue-specific,light-regulated and plastid-regulated expression of the single-copy nuclear gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana

The single-copy PetC gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana consists of five exons interrupted by four introns and encodes a protein of 229 amino acid residues with extensive sequence similarity to the chloroplast Rieske proteins of other higher plants. The N-terminal 50 amino acid residues constitute a presequence for targeting to the chloroplast and the remaining 179 amino acid residues make up the mature protein. Three of the introns are in identical positions in the PetC gene of Chlamydomonas reinhardtii, suggesting that they are of ancient origin. RNA-blot hybridisation showed that the gene was expressed in shoots, but not roots, and was light regulated and repressed by sucrose. The expression of chimeric genes consisting of PetC promoter fragments fused to the beta-glucuronidase (GUS) reporter gene was examined in A. thaliana and tobacco. In A. thaliana, GUS activity was detected in leaves, stems, flowers and siliques, but not in roots, and showed a strong correlation with the presence of chloroplasts. In transgenic tobacco, low levels of GUS activity were also detected in light-exposed roots. GUS activity in transgenic tobacco seedlings was light regulated and was decreased by norflurazon in the light suggesting regulation of PetC expression by plastid signals.     

A new gun mutant of oilseed rape with a reduced porphyrin flux through Mg-chelatase

Plastid-to-nucleus retrograde signaling coordinates the expression of nuclear photosynthetic genes with the developmental and functional state of the plastid. These signals are essential not only for coordinating the expression of photosynthetic genes both in the plastome and nuclear genome, but also for plants to respond optimally to environmental stress. In the present study, we found that the expression of the nuclear genes that encode plastid and non-plastid photosynthesis-related proteins was still maintained or slightly higher in cr3529, a chlorophyll deficient mutant of oilseed rape that possesses an arrested development of chloroplasts, suggesting that the expression of photosynthesis-related nuclear genes was uncoupled from the normal dependence on the developmental state of the chloroplast in cr3529. When the development of the plastid in cr3529 and the wild type was completely inhibited by lincomycin, much higher expression of photosynthesis-related nuclear genes was observed in cr3529, suggesting that the genomes uncoupled (gun) phenotype of cr3529 is even more apparent than under normal growth conditions. Lincomycin treatment also derepressed the expression of plastid genes in cr3529. The determination of porphyrin flux through Mg-chelatase showed that the content of protoporphyrin IX and Mg-protoporphyrin decreased in cr3529. The obvious gun phenotype of cr3529 under normal growth conditions and the pattern of tetrapyrrole metabolism in cr3529 suggest that it is a new gun mutant that could be used to study the regulation of the expression of nuclear and plastid genes by plastid-to-nucleus retrograde signaling under more physiological conditions and the mechanism of plant stress responses mediated by plastid signals.     

An Arabidopsis cotyledon-specific albino locus: a possible role in 16S rRNA maturation

We report here the isolation and characterization of a cotyledon-specific albino locus of Arabidopsis, WHITE COTYLEDONS (WCO). This recessive mutation in the WCO locus, located on the top of Chromosome 1, results in albino cotyledons but green true leaves. An accumulation profile of chlorophylls and ultrastructure of chloroplasts indicate that WCO is necessary for development of functional chloroplasts in cotyledons but is dispensable in true leaves. This was further supported by the fact that the mutants request feeding of sucrose for their survival at the early seedling stage where true leaves have not emerged, but the mutants which have developed true leaves are able to grow autotrophically without sucrose supplementation. The wco mutants accumulate low levels of chloroplast mRNA encoding photosynthesis-related proteins and have a specific defect in 16S rRNA maturation in a cotyledon-specific manner. Although wco mutants exhibited abnormal chloroplasts and chloroplast gene expression in cotyledons, nuclear genes for photosynthetic components are expressed at similar levels to those found in wild-type siblings. This lack of suppression of the nuclear genes is not due to a defect in the signaling of the so-called "plastid factor" to the nucleus since normal suppression of the nuclear genes was observed in response to the photo-oxidative damage due to norflurazon application.     

PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts

Summary Evidence is presented for the introduction of functional copies of the GUS-reporter gene with plastid regulatory signals into chloroplasts after treatment of Nicotiana plumbaginifolia leaf protoplasts with PEG. GUS-activity is found in cells derived from protoplasts treated with PEG in the presence of plasmids harbouring the GUS-gene under the control of plastid promoter and terminator signals (plastid-specific reporter gene constructions). The activity is maintained after chloroplast isolation and incubation with the protease thermolysin under conditions sufficient to completely remove the much higher transient nuclear/cytoplasmic expression of a GUS-gene carrying the CaMV 35S-promoter. Likewise, GUS-activity derived from a plasmid coding for the nuclear/cytoplasmic expression of the reporter gene with a plastid transit presequence is also maintained after these procedures. These results indicate that PEG-treatment is a suitable protocol by which to introduce DNA into chloroplasts for the study of transient gene expression.     

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor sigma B

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.     

Chloroplastic regulation of apoplastic alpha-amylase activity in pea seedlings

Photobleaching of pea (Pisum sativum L.) seedling leaves by treatment with norflurazon (San 9789) and 7 days of continuous white light caused a 76- to 85-fold increase in the activity of the primary α-amylase, a largely apoplastic enzyme, over normally greening seedlings. Levels of chlorophyll were near zero and levels of plastid marker enzyme activities were very low in norflurazon-treated seedlings, indicating severe photooxidative damage to plastids. As levels of norflurazon or fluence rates were lowered, decreasing photobleaching of tissues, α-amylase activity decreased. Levels of leaf β-amylase and starch debranching enzyme changed very little in norflurazon-treated seedlings. Infiltration extraction of leaves of norflurazon-treated and normally greening seedlings indicated that at least 57 and 62%, respectively, of α-amylase activity was in the apoplast. α-Amylase activity recovered from the apoplast of photobleached leaves of norflurazon-treated seedlings was 18-fold higher than that for green leaves. Inhibitors of photosynthesis (DCMU and atrazine) and an inhibitor of chlorophyll accumulation that does not cause photooxidation of plastid components (tentoxin) had little effect on levels of α-amylase activity, indicating norflurazon-caused loss of chlorophyll and lack of photosynthesis did not cause the large induction in α-amylase activity. An inhibitor of both abscisic acid and gibberellin synthesis (paclobutrazol [PP333]) and an analog of norflurazon which inhibits photosynthesis but not carotenoid synthesis (San 9785) caused only moderate (about five-fold) increases in α-amylase activity. Lincomycin and chloramphenicol increased α-amylase activity in light grown seedings to the same magnitude as norflurazon, indicating that the effect of norflurazon is probably through the destruction of plastid ribosomes. It is proposed that chloroplasts produce a negative signal for the regulation of the apoplastic α-amylase in pea.     

The discovery of plastid-to-nucleus retrograde signaling—a personal perspective

DNA and machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is relatively small, that most genes for chloroplast-localized proteins reside in the nucleus and that chloroplast membranes, ribosomes, and protein complexes are composed of proteins encoded in both the chloroplast and the nuclear genome. This situation has made the existence of mechanisms highly probable that coordinate the gene expression in plastids and nucleus. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided by studies on plastid ribosome deficient mutants with reduced amounts and/or activities of nuclear-encoded chloroplast proteins including the small subunit of Rubisco, ferredoxin NADP+ reductase, and enzymes of the Calvin cycle. This review describes first models of plastid-to-nucleus signaling and their discovery. Today, many plastid signals are known. They do not only balance gene expression in chloroplasts and nucleus during developmental processes but are also generated in response to environmental changes sensed by the organelles.