Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information

SUMMARYA lack of individual plastid ribosomal proteins (PRPs) can have diverse phenotypic effects in Arabidopsis thaliana, ranging from embryo lethality to compromised vitality, with the latter being associated with photosynthetic lesions and decreases in the expression of plastid proteins. In this study, reverse genetics was employed to study the function of eight PRPs, five of which (PRPS1, -S20, -L27, -L28 and -L35) have not been functionally characterised before. In the case of PRPS17, only leaky alleles or RNA interference lines had been analysed previously. PRPL1 and PRPL4 have been described as essential for embryo development, but their mutant phenotypes are analysed in detail here. We found that PRPS20, -L1, -L4, -L27 and -L35 are required for basal ribosome activity, which becomes crucial at the globular stage and during the transition from the globular to the heart stage of embryogenesis. Thus, lack of any of these PRPs leads to alterations in cell division patterns, and embryo development ceases prior to the heart stage. PRPL28 is essential at the latest stages of embryo-seedling development, during the greening process. PRPS1, -S17 and -L24 appear not to be required for basal ribosome activity and the organism can complete its entire life cycle in their absence. Interestingly, despite the prokaryotic origin of plastids, the significance of individual PRPs for plant development cannot be predicted from the relative phenotypic severity of the corresponding mutants in prokaryotic systems.

Plastid gene expression is crucial for organelle function, but the factors that control it are still largely unclear. Members of the socalled mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate organellar gene expression at different levels. Arabidopsis (Arabidopsis thaliana) mTERF6 is localized in chloroplasts and mitochondria, and its knockout perturbs plastid development and results in seedling lethality. In the leaky mterf6-1 mutant, a defect in photosynthesis is associated with reduced levels of photosystem subunits, although corresponding messenger RNA levels are unaffected, whereas translational capacity and maturation of chloroplast ribosomal RNAs (rRNAs) are perturbed in mterf6-1 mutants. Bacterial one-hybrid screening, electrophoretic mobility shift assays, and coimmunoprecipitation experiments reveal a specific interaction between mTERF6 and an RNA sequence in the chloroplast isoleucine transfer RNA gene (trnI.2) located in the rRNA operon. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. At present, it is unclear whether disturbed rRNA maturation is a primary or secondary defect. However, it is clear that mTERF6 is required for the maturation of trnI.2. This points to an additional function of mTERFs.

Plants need tight regulation of photosynthetic electron transport for survival and growth under environmental and metabolic conditions. For this purpose, the linear electron transport (LET) pathway is supplemented by a number of alternative electron transfer pathways and valves. In Arabidopsis, cyclic electron transport (CET) around photosystem I (PSI), which recycles electrons from ferrodoxin to plastoquinone, is the most investigated alternative route. However, the interdependence of LET and CET and the relative importance of CET remain unclear, largely due to the difficulties in precise assessment of the contribution of CET in the presence of LET, which dominates electron flow under physiological conditions. We therefore generated Arabidopsis mutants with a minimal water-splitting activity, and thus a low rate of LET, by combining knockout mutations in PsbO1, PsbP2, PsbQ1, PsbQ2, and PsbR loci. The resulting Δ5 mutant is viable, although mature leaves contain only ∼ 20% of wild-type naturally less abundant PsbO2 protein. Δ5 plants compensate for the reduction in LET by increasing the rate of CET, and inducing a strong non-photochemical quenching (NPQ) response during dark-to-light transitions. To identify the molecular origin of such a high-capacity CET, we constructed three sextuple mutants lacking the qE component of NPQ (Δ5 npq4-1), NDH-mediated CET (Δ5 crr4-3), or PGR5-PGRL1-mediated CET (Δ5 pgr5). Their analysis revealed that PGR5-PGRL1-mediated CET plays a major role in ΔpH formation and induction of NPQ in C3 plants. Moreover, while pgr5 dies at the seedling stage under fluctuating light conditions, Δ5 pgr5 plants are able to survive, which underlines the importance of PGR5 in modulating the intersystem electron transfer.

Summary Correct chloroplast development and function require co‐ordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast‐localized pentatricopeptide repeat (PPR) protein with a C‐terminal Small MutS‐Related (SMR) domain, is involved in integrating multiple developmental and stress‐related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signalling pathways. Here we show that following perturbation of chloroplast protein homeostasis: (i) by growth in lincomycin‐containing medium; or (ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21‐1 and prpl11‐1) or plastid protein import and folding (cphsc70‐1), GUN1 influences NEP‐dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.

These authors contributed equally to this work. SUMMARYThe oxygen-evolving complex of eukaryotic photosystem II (PSII) consists of four extrinsic subunits, PsbO (33 kDa), PsbP (23 kDa), PsbQ (17 kDa) and PsbR (10 kDa), encoded by seven nuclear genes, PsbO1 (At5g66570), PsbO2 (At3g50820), PsbP1 (At1g06680), PsbP2 (At2g30790), PsbQ1 (At4g21280), PsbQ2 (At4g05180) and PsbR (At1g79040). Using Arabidopsis insertion mutant lines, we show that PsbP1, but not PsbP2, is essential for photoautotrophic growth, whereas plants lacking both forms of PsbQ and/or PsbR show normal growth rates. Complete elimination of PsbQ has a minor effect on PSII function, but plants lacking PsbR or both PsbR and PsbQ are characterized by more pronounced defects in PSII activity. Gene expression and immunoblot analyses indicate that accumulation of each of these proteins is highly dependent on the presence of the others, and is controlled at the post-transcriptional level, whereas PsbO stability appears to be less sensitive to depletion of other subunits of the oxygen-evolving complex. In addition, comparison of levels of the PSII super-complex in wild-type and mutant leaves reveals the importance of the individual subunits of the oxygen-evolving complex for the supramolecular organization of PSII and their influence on the rate of state transitions.

DEAD-box RNA helicases (DBRHs) modulate RNA secondary structure, allowing RNA molecules to adopt the conformations required for interaction with their target proteins. RH50 is a chloroplast-located DBRH that colocalizes and is coexpressed with GUN1, a central factor in chloroplast-to-nucleus signaling. When combined with mutations that impair plastid gene expression (prors1-1, prpl11-1, prps1-1, prps21-1, prps17-1, and prpl24-1), rh50 and gun1 mutations evoke similar patterns of epistatic effects. These observations, together with the synergistic growth phenotype of the double mutant rh50-1 gun1-102, suggest that RH50 and GUN1 are functionally related and that this function is associated with plastid gene expression, in particular ribosome functioning. However, rh50-1 itself is not a gun mutant, although-like gun1-102-the rh50-1 mutation suppresses the downregulation of nuclear genes for photosynthesis induced by the prors1-1 mutation. The RH50 protein comigrates with ribosomal particles, and is required for efficient translation of plastid proteins. RH50 binds to transcripts of the 23S-4.5S intergenic region and, in its absence, levels of the corresponding rRNA processing intermediate are strongly increased, implying that RH50 is required for the maturation of the 23S and 4.5S rRNAs. This inference is supported by the finding that loss of RH50 renders chloroplast protein synthesis sensitive to erythromycin and exposure to cold. Based on these results, we conclude that RH50 is a plastid rRNA maturation factor.

The drought–stress response in plant involves the cross-talk between abscisic acid (ABA) and other phytohormones, such as jasmonates and ethylene. The auxin indole-3-acetic acid (IAA) plays an integral part in plant adaptation to drought stress. Investigation was made to see how the main auxin IAA interacted with other plant hormones under water stress, applied through two different growth conditions (solid and hydroponic). Medicago sativa plants nodulated by the Ensifer meliloti wild type 1021 (Ms-1021) and its IAA-overproducing RD64 derivative strains (Ms-RD64) were subjected to drought stress, comparing their response. When the expression of nifH gene and the activity of the nitrogenase enzyme were measured after stress treatments, Ms-RD64 plants recorded a significantly weaker damage. These results were correlated with a lower biomass reduction, and a higher Rubisco protein level measured for the Ms-RD64-stressed plants as compared to the Ms-1021-stressed ones. It has been verified that the stress response observed for Ms-RD64-stressed plants was related to the production of greater amount of low-molecular-weight osmolytes, such as proline and pinitol, measured in these plants. For the Ms-RD64 plants the immunoblotting analysis of thylakoid membrane proteins showed that some of the photosystem proteins increased after the stress. An increased non-photochemical quenching after the stress was also observed for these plants. The reduced wilting signs observed for these plants were also connected to the significant down-regulation of the MtAA03 gene involved in the ABA biosynthesis, and with the unchanged expression of the two genes (Mt-2g006330 and Mt-8g095330) of ABA signaling. When the expression level of the ethylene-signaling genes was evaluated by qPCR analysis no significant alteration of the key positive regulators was recorded for Ms-RD64-stressed plants. Coherently, these plants accumulated 40% less ethylene as compared to Ms-1021-stressed ones. The results presented herein indicate that the variations in endogenous IAA levels, triggered by the overproduction of rhizobial IAA inside root nodules, positively affected drought stress response in nodulated alfalfa plants.