Cytokinin Regulates the Etioplast-Chloroplast Transition through the Two-Component Signaling System and Activation of Chloroplast-Related Genes.

One of the classical functions of the plant hormone cytokinin is the regulation of plastid development, but the underlying molecular mechanisms remain elusive. In this study, we employed a genetic approach to evaluate the role of cytokinin and its signaling pathway in the light-induced development of chloroplasts from etioplasts in Arabidopsis (Arabidopsis thaliana). Cytokinin increases the rate of greening and stimulates ultrastructural changes characteristic for the etioplast-to-chloroplast transition. The steady-state levels of metabolites of the tetrapyrrole biosynthesis pathway leading to the production of chlorophyll are enhanced by cytokinin. This effect of cytokinin on metabolite levels arises due to the modulation of expression for chlorophyll biosynthesis genes such as HEMA1, GUN4, GUN5, and CHLM Increased expression of HEMA1 is reflected in an enhanced level of the encoded glutamyl-tRNA reductase, which catalyzes one of the rate-limiting steps of chlorophyll biosynthesis. Mutant analysis indicates that the cytokinin receptors ARABIDOPSIS HIS KINASE2 (AHK2) and AHK3 play a central role in this process. Furthermore, the B-type ARABIDOPSIS RESPONSE REGULATOR1 (ARR1), ARR10, and ARR12 play an important role in mediating the transcriptional output during etioplast-chloroplast transition. B-type ARRs bind to the promotors of HEMA1 and LHCB6 genes, indicating that cytokinin-dependent transcription factors directly regulate genes of chlorophyll biosynthesis and the light harvesting complex. Together, these results demonstrate an important role for the cytokinin signaling pathway in chloroplast development, with the direct transcriptional regulation of chlorophyll biosynthesis genes as a key aspect for this hormonal control.

Arabidopsis chlorophyll biosynthesis: an essential balance between the methylerythritol phosphate and tetrapyrrole pathways.

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)-derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis.

The GUN4 protein plays a regulatory role in tetrapyrrole biosynthesis and chloroplast-to-nucleus signalling in Chlamydomonas reinhardtii.

The GENOMES UNCOUPLED 4 (GUN4) protein is found only in aerobic photosynthetic organisms. We investigated the role of GUN4 in metabolic activities of the Mg branch of the tetrapyrrole biosynthesis pathway and the plastid signal-mediated changes of nuclear gene expression in Chlamydomonas reinhardtii. In light, gun4 accumulates only 40% of the wild-type chlorophyll level. Light- or dark-grown gun4 mutant accumulates high levels of protoporphyrin IX (Proto), and displays increased sensitivity to moderate light intensities. Despite the photooxidative stress, gun4 fails to downregulate mRNA levels of the tetrapyrrole biosynthesis and the photosynthesis-associated nuclear genes (PhANGs). In contrast, upon illumination, the Proto-accumulating and light-sensitive chlD-1 mutant displays the expected downregulation of the same nuclear genes. Although chlD-1 and the wild type have similar GUN4 transcript levels, the GUN4 protein in chlD-1 is hardly detectable. Overexpression of GUN4 in chlD-1 modifies the downregulation of nuclear gene expression, but also increases light tolerance. Therefore, GUN4 is proposed to function in 'shielding' Proto, and most likely MgProto, by reducing reactivity with O2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to (1) O2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.

A Shoot-Specific Hypoxic Response of Arabidopsis Sheds Light on the Role of the Phosphate-Responsive Transcription Factor PHOSPHATE STARVATION RESPONSE1.

Plant responses to biotic and abiotic stresses are often very specific, but signal transduction pathways can partially or completely overlap. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the transcriptional responses to phosphate starvation and oxygen deficiency stress comprise a set of commonly induced genes. While the phosphate deficiency response is systemic, under oxygen deficiency, most of the commonly induced genes are found only in illuminated shoots. This jointly induced response to the two stresses is under control of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), but not of the oxygen-sensing N-end rule pathway, and includes genes encoding proteins for the synthesis of galactolipids, which replace phospholipids in plant membranes under phosphate starvation. Despite the induction of galactolipid synthesis genes, total galactolipid content and plant survival are not severely affected by the up-regulation of galactolipid gene expression in illuminated leaves during hypoxia. However, changes in galactolipid molecular species composition point to an adaptation of lipid fluxes through the endoplasmic reticulum and chloroplast pathways during hypoxia. PHR1-mediated signaling of phosphate deprivation was also light dependent. Because a photoreceptor-mediated PHR1 activation was not detectable under hypoxia, our data suggest that a chloroplast-derived retrograde signal, potentially arising from metabolic changes, regulates PHR1 activity under both oxygen and phosphate deficiency.

Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability.

The consequences of ketocarotenoid production in transgenic tobacco (Nicotiana tabacum) plants expressing a Chlamydomonas reinhardtii gene encoding a ß-carotene ketolase were examined concerning the functionality of the photosynthetic apparatus. T1 plants produced less photosynthetic pigments per dry weight, but Chl a/Chl b ratios remained unchanged. Almost as much ketocarotenoids as accessory xanthophylls accumulated per Chl a molecule. These ketocarotenoids were found mainly in the thylakoid membranes, but were not functionally bound to light-harvesting complexes, although LHCII is known to be able to bind astaxanthin. On the contrary, high amounts of ketocarotenoids probably changed the properties of the lipid phase of the thylakoids, thereby reducing the stability of photosystem II supercomplexes and LHCII trimers and ultimately decreasing grana formation. In addition, photosystem II function in electron transport was impaired, and plants exhibited less non-photochemical quenching compared to wild-type plants. Thus, in order not to disturb vital functions of the plants, production of astaxanthin and other nutritionally valuable ketocarotenoids apparently requires ways to sequester the additional carotenoids to plastoglobuli.

Functional characterization of the two ferrochelatases in Arabidopsis thaliana.

The enzyme ferrochelatase catalyses the formation of protoheme by inserting Fe(2+) into protoporphyrin IX. Although most organisms express only one ferrochelatase, all land plants analysed so far possess at least two ferrochelatase proteins. Analysis of publicly available expression data suggests that the two Arabidopsis thaliana ferrochelatases, FC1 and FC2, serve different functions, corroborating previous assumptions. Co-expression analysis of FC1 and FC2, together with microarray analyses, implies that fc1 and fc2 trigger different modes of plastid signalling in roots and leaves, respectively, and indicates that FC2 might be involved in stress responses. Thus, loss of FC2 increases resistance to salt and flagellin treatment. Whereas fc1 plants showed no obvious mutant phenotype, fc2 mutants formed abnormally small, pale green rosette leaves; were low in chlorophylls, carotenoids and several photosynthetic proteins; and their photosynthetic performance was impaired. These phenotypes are attenuated by growth in continuous light, in agreement with the finding that fc2 plants accumulate protochlorophyllide and display a fluorescent (flu) phenotype in the dark. In consequence we show that, contrary to earlier suggestions, FC2 produces heme not only for photosynthetic cytochromes, but also for proteins involved in stress responses, whereas the impairment of FC1 apparently interferes only marginally with stress responses.

Posttranslational influence of NADPH-dependent thioredoxin reductase C on enzymes in tetrapyrrole synthesis.

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steady-state levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts.

Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation (redox) state of the photosynthetic electron chain that acts as a trigger for compensatory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes (ERGs) in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes.

Induced deactivation of genes encoding chlorophyll biosynthesis enzymes disentangles tetrapyrrole-mediated retrograde signaling.

In photosynthetic organisms, tetrapyrrole-mediated retrograde signals are proposed to contribute to a balanced nuclear gene expression (NGE) in response to metabolic activity in chloroplasts. We followed an experimental short-term approach that allowed the assessment of modified NGE during the first hours of specifically modified enzymatic steps of the Mg branch of tetrapyrrole biosynthesis, when pleiotropic effects of other signals can be avoided. In response to 24-h-induced silencing of CHLH, CHLM, and CHL27 encoding the CHLH subunit of Mg chelatase, the Mg protoporphyrin methyltransferase and Mg protoporphyrin monomethylester cyclase, respectively, deactivated gene expression rapidly led to reduced activity of the corresponding enzymes and altered Mg porphyrin levels. But NGE was not substantially altered. When these three genes were continuously inactivated for up to 4 d, changes of transcript levels of nuclear genes were determined. CHL27 silencing for more than 24h results in necrotic leaf lesions and modulated transcript levels of oxidative stress-responsive and photosynthesis-associated nuclear genes (PhANGs). The prolonged deactivation of CHLH and CHLM results in slightly elevated transcript levels of PhANGs and tetrapyrrole-associated genes. These time-resolved studies indicate a complex scenario for the contribution of tetrapyrrole biosynthesis on NGE mediated by (1)O2-induced signaling and feedback-regulated ALA synthesis.

Virus-induced gene silencing of pea CHLI and CHLD affects tetrapyrrole biosynthesis, chloroplast development and the primary metabolic network.

The first committed and highly regulated step of chlorophyll biosynthesis is the insertion of Mg(2+) into protoporphyrin IX, which is catalyzed by Mg chelatase that consists of CHLH, CHLD and CHLI subunits. In this study, CHLI and CHLD genes were suppressed by virus-induced gene silencing (VIGS-CHLI and VIGS-CHLD) in pea (Pisum sativum), respectively. VIGS-CHLI and VIGS-CHLD plants both showed yellow leaf phenotypes with the reduced Mg chelatase activity and the inactivated synthesis of 5-aminolevulinic acid. The lower chlorophyll accumulation correlated with undeveloped thylakoid membranes, altered chloroplast nucleoid structure, malformed antenna complexes and compromised photosynthesis capacity in the yellow leaf tissues of the VIGS-CHLI and VIGS-CHLD plants. Non-enzymatic antioxidant contents and the activities of antioxidant enzymes were altered in response to enhanced accumulation of reactive oxygen species (ROS) in the chlorophyll deficient leaves of VIGS-CHLI and VIGS-CHLD plants. Furthermore, the results of metabolite profiling indicate a tight correlation between primary metabolic pathways and Mg chelatase activity. We also found that CHLD induces a feedback-regulated change of the transcription of photosynthesis-associated nuclear genes. CHLD and CHLI silencing resulted in a rapid reduction of photosynthetic proteins. Taken together, Mg chelatase is not only a key regulator of tetrapyrrole biosynthesis but its activity also correlates with ROS homeostasis, primary interorganellar metabolism and retrograde signaling in plant cells.