GUN control in retrograde signaling: How GENOMES UNCOUPLED proteins adjust nuclear gene expression to plastid biogenesis.

Communication between cellular compartments is vital for development and environmental adaptation. Signals emanating from organelles, so-called retrograde signals, coordinate nuclear gene expression with the developmental stage and/or the functional status of the organelle. Plastids (best known in their green photosynthesizing differentiated form, the chloroplasts) are the primary energy-producing compartment of plant cells, and the site for the biosynthesis of many metabolites, including fatty acids, amino acids, nucleotides, isoprenoids, tetrapyrroles, vitamins, and phytohormone precursors. Signals derived from plastids regulate the accumulation of a large set of nucleus-encoded proteins, many of which localize to plastids. A set of mutants defective in retrograde signaling (genomes uncoupled, or gun) was isolated over 25 years ago. While most GUN genes act in tetrapyrrole biosynthesis, resolving the molecular function of GUN1, the proposed integrator of multiple retrograde signals, has turned out to be particularly challenging. Based on its amino acid sequence, GUN1 was initially predicted to be a plastid-localized nucleic acid-binding protein. Only recently, mechanistic information on the function of GUN1 has been obtained, pointing to a role in plastid protein homeostasis. This review article summarizes our current understanding of GUN-related retrograde signaling and provides a critical appraisal of the various proposed roles for GUNs and their respective pathways.

Super-Transparent Soil for In Situ Observation of Root Phenotypes.

Transparent soil (TS) presents immense potential for root phenotyping due to its ability to facilitate high-resolution imaging. However, challenges related to transparency, mechanical properties, and cost hinder its development. Herein, we introduce super-transparent soil (s-TS) prepared via the droplet method using low acyl gellan gum and hydroxyethyl cellulose crosslinked with magnesium ions. The refractive index of the hydroxyethyl cellulose solution (1.345) closely aligns with that of water (1.333) and the low acyl gellan gum solution (1.340), thereby significantly enhancing the transmittance of hydrogel-based transparent soil. Optimal transmittance (98.45%) is achieved with polymer concentrations ranging from 0.8 to 1.6 wt.% and ion concentrations between 0.01 and 0.09 mol·L-1. After 60 days of plant cultivation, s-TS maintains a transmittance exceeding 89.5%, enabling the detailed visualization of root growth dynamics. Furthermore, s-TS exhibits remarkable mechanical properties, withstanding a maximum compressive stress of 477 kPa and supporting a maximum load-bearing depth of 186 cm. This innovative approach holds promising implications for advanced root phenotyping studies, fostering the investigation of root heterogeneity and the development of selective expression under controlled conditions.

Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals.

Retrograde signals emanate from the DNA-containing cell organelles (plastids and mitochondria) and control the expression of a large number of nuclear genes in response to environmental and developmental cues. Previous studies on retrograde signaling have mainly analyzed the regulation of nuclear gene expression at the transcript level. To determine the contribution of translational and posttranslational regulation to plastid retrograde signaling, we combined label-free proteomics with transcriptomic analysis of Arabidopsis (Arabidopsis thaliana) seedlings and studied their response to interference with the plastid gene expression pathway of retrograde signaling. By comparing the proteomes of the genomes uncoupled1 (gun1) and gun5 mutants with the wild type, we show that GUN1 is critical in the maintenance of plastid protein homeostasis (proteostasis) when plastid translation is blocked. Combining transcriptomic and proteomic analyses of the wild type and gun1, we identified 181 highly translationally or posttranslationally regulated (HiToP) genes. We demonstrate that HiToP photosynthesis-associated nuclear genes (PhANGs) are largely regulated by translational repression, while HiToP ribosomal protein genes are regulated posttranslationally, likely at the level of protein stability without the involvement of GUN1. Our findings suggest distinct posttranscriptional control mechanisms of nuclear gene expression in response to plastid-derived retrograde signals. They also reveal a role for GUN1 in the translational regulation of several PhANGs and highlight extensive posttranslational regulation that does not necessitate GUN1. This study advances our understanding of the molecular mechanisms underlying intracellular communication and provides new insight into cellular responses to impaired plastid protein biosynthesis.

Control of Retrograde Signaling by Rapid Turnover of GENOMES UNCOUPLED1.

The exchange of signals between cellular compartments coordinates development and differentiation, modulates metabolic pathways, and triggers responses to environmental conditions. The proposed central regulator of plastid-to-nucleus retrograde signaling, GENOMES UNCOUPLED1 (GUN1), is present at very low levels, which has hampered the discovery of its precise molecular function. Here, we show that the Arabidopsis (Arabidopsis thaliana) GUN1 protein accumulates to detectable levels only at very early stages of leaf development, where it functions in the regulation of chloroplast biogenesis. GUN1 mRNA is present at high levels in all tissues, but GUN1 protein undergoes rapid degradation (with an estimated half-life of ∼4 h) in all tissues where chloroplast biogenesis has been completed. The rapid turnover of GUN1 is controlled mainly by the chaperone ClpC1, suggesting degradation of GUN1 by the Clp protease. Degradation of GUN1 slows under stress conditions that alter retrograde signaling, thus ensuring that the plant has sufficient GUN1 protein. We also find that the pentatricopeptide repeat motifs of GUN1 are important determinants of GUN1 stability. Moreover, overexpression of GUN1 causes an early flowering phenotype, suggesting a function of GUN1 in developmental phase transitions beyond chloroplast biogenesis. Taken together, our results provide new insight into the regulation of GUN1 by proteolytic degradation, uncover its function in early chloroplast biogenesis, and suggest a role in developmental phase transitions.

High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves.

KEY MESSAGE: The potent anti-HIV microbicide griffithsin was expressed to high levels in tobacco chloroplasts, enabling efficient purification from both fresh and dried biomass, thus providing storable material for inexpensive production and scale-up on demand. The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand.

Functional conservation of GENOMES UNCOUPLED1 in plastid-to-nucleus retrograde signaling in tomato.

Retrograde signaling between plastids and the nucleus is vital for chloroplast biogenesis and environmental responses. GENOMES UNCOUPLED1 (GUN1) was proposed to be a central integrator of multiple retrograde signaling pathways in the model plant Arabidopsis thaliana (Arabidopsis). However, the function of GUN1 orthologs in other plant species has not been well studied. Here, we found that many GUN1 orthologs from the Solanaceae family have a short N-terminus before the first pentatricopeptide repeat (PPR) motif which is predicted as intrinsically disordered regions (IDRs). Functional analyses of tomato (Solanum lycopersicum L.) GUN1 (SlGUN1), which does not contain N-terminal IDRs, show that it can complement the GUN phenotype of the Arabidopsis gun1 mutant (Atgun1). However, in contrast to the AtGUN1 protein, which does contain the N-terminal IDRs, the SlGUN1 protein is highly accumulated even after chloroplast biogenesis is completed, suggesting that the N-terminal IDRs may determine the stability of the GUN1 protein. Furthermore, we generated tomato Slgun1 genome-edited mutants via the CRISPR-Cas9 system. The Slgun1 mutants exhibited a typical GUN phenotype under lincomycin (Lin) or norflurazon (NF) treatment. Moreover, Slgun1 mutants are hypersensitive to low concentrations of Lin or NF. Taken together, our results suggest that, although lacking the N-terminal IDRs, SlGUN1 plays conserved roles in plastid retrograde signaling in tomato plants.

Arabidopsis ß-ketoacyl-[acyl carrier protein] synthase i is crucial for fatty acid synthesis and plays a role in chloroplast division and embryo development.

Lipid metabolism plays a pivotal role in cell structure and in multiple plant developmental processes. ß-Ketoacyl-[acyl carrier protein] synthase I (KASI) catalyzes the elongation of de novo fatty acid (FA) synthesis. Here, we report the functional characterization of KASI in the regulation of chloroplast division and embryo development. Phenotypic observation of an Arabidopsis thaliana T-DNA insertion mutant, kasI, revealed multiple morphological defects, including chlorotic (in netted patches) and curly leaves, reduced fertility, and semidwarfism. There are only one to five enlarged chloroplasts in the mesophyll cells of chlorotic sectors of young kasI rosette leaves, indicating suppressed chloroplast division under KASI deficiency. KASI deficiency results in a significant change in the polar lipid composition, which causes the suppressed expression of FtsZ and Min system genes, disordered Z-ring placement in the oversized chloroplast, and inhibited polymerization of FtsZ protein at mid-site of the chloroplast in kasI. In addition, KASI deficiency results in disrupted embryo development before the globular stage and dramatically reduces FA levels (~33.6% of the wild type) in seeds. These results demonstrate that de novo FA synthesis is crucial and has pleiotropic effects on plant growth. The polar lipid supply is important for chloroplast division and development, revealing a key function of FA synthesis in plastid development.

Chloroplast proteostasis: A story of birth, life, and death.

Protein homeostasis (proteostasis) is a dynamic balance of protein synthesis and degradation. Because of the endosymbiotic origin of chloroplasts and the massive transfer of their genetic information to the nucleus of the host cell, many protein complexes in the chloroplasts are constituted from subunits encoded by both genomes. Hence, the proper function of chloroplasts relies on the coordinated expression of chloroplast- and nucleus-encoded genes. The biogenesis and maintenance of chloroplast proteostasis are dependent on synthesis of chloroplast-encoded proteins, import of nucleus-encoded chloroplast proteins from the cytosol, and clearance of damaged or otherwise undesired "old" proteins. This review focuses on the regulation of chloroplast proteostasis, its interaction with proteostasis of the cytosol, and its retrograde control over nuclear gene expression. We also discuss significant issues and perspectives for future studies and potential applications for improving the photosynthetic performance and stress tolerance of crops.

Shanghai RAPESEED Database: a resource for functional genomics studies of seed development and fatty acid metabolism of Brassica.

The Shanghai RAPESEED Database (RAPESEED, http://rapeseed.plantsignal.cn/) was created to provide the solid platform for functional genomics studies of oilseed crops with the emphasis on seed development and fatty acid metabolism. The RAPESEED includes the resource of 8462 unique ESTs, of which 3526 clones are with full length cDNA; the expression profiles of 8095 genes and the Serial Analysis of Gene Expression (SAGE, 23,895 unique tags) and tag-to-gene data during seed development. In addition, a total of approximately 14,700 M3 mutant populations were generated by ethylmethanesulfonate (EMS) mutagenesis and related seed quality information was determined using the Foss NIR System. Further, the TILLING (Targeting Induced Local Lesions IN Genomes) platform was established based on the generated EMS mutant population. The relevant information was collected in RAPESEED database, which can be searched through keywords, nucleotide or protein sequences, or seed quality parameters, and downloaded.

Control of retrograde signalling by protein import and cytosolic folding stress.

Communication between organelles and the nucleus is essential for fitness and survival. Retrograde signals are cues emitted from the organelles to regulate nuclear gene expression. GENOMES UNCOUPLED1 (GUN1), a protein of unknown function, has emerged as a central integrator, participating in multiple retrograde signalling pathways that collectively regulate the nuclear transcriptome. Here, we show that GUN1 regulates chloroplast protein import through interaction with the import-related chaperone cpHSC70-1. We demonstrated that overaccumulation of unimported precursor proteins (preproteins) in the cytosol causes a GUN phenotype in the wild-type background and enhances the GUN phenotype of the gun1 mutant. Furthermore, we identified the cytosolic HSP90 chaperone complex, induced by overaccumulated preproteins, as a central regulator of photosynthetic gene expression that determines the expression of the GUN phenotype. Taken together, our results suggest a model in which protein import capacity, folding stress and the cytosolic HSP90 complex control retrograde communication.

Synthesis and evaluation of novel prodrugs of foscarnet and dideoxycytidine with a universal carrier compound comprising a chemiluminescent and a photochromic conjugate.

To facilitate intracellular delivery of hydrophilic drugs, a general lipophilic carrier molecule was designed and synthesized. The carrier comprised a chemiluminescent-photochromic conjugate that potentiates diffusion across cell membranes to enhance intracellular uptake of the drug. The designed mechanism involves activation of the chemiluminescent moiety by intracellular oxygen free radicals and intermolecular energy transfer of the excited state energy to the photochromic moiety to result in release of the drug to allow the desired pharmacological effect to occur. Prodrugs of foscarnet and dideoxycytidine with several carriers caused suppression of a human immunodeficiency virus infection in human cultured macrophages that was up to five times more effective than the drug alone. Successful in vivo efficacy testing of prodrug has been accomplished by demonstrating the suppression of a retroviral infection of Friend leukemia virus in mice. Acute toxicity studies of the carrier indicated that it was nontoxic.

Global analysis of gene expression profiles in Brassica napus developing seeds reveals a conserved lipid metabolism regulation with Arabidopsis thaliana.

In order to study Brassica napus fatty acid (FA) metabolism and relevant regulatory networks, a systematic identification of fatty acid (FA) biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization (>8000 EST clones from seed). The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17-21 d after flowering (DAF), whereas FA biosynthesis-related genes were highly expressed primarily at 21 DAF. Hormone (auxin and jasmonate) signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.