'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.     

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.

This review summarizes new insights in GUN-mediated retrograde signaling, and highlights outstanding questions and challenges that should be addressed in future research.     

Plant signaling systems. Plastid-generated signals and their role in nuclear gene expression

Current concepts are outlined regarding the chloroplast effects on expression of the nuclear genes encoding plastid proteins. The major types of plastid-generated signals are considered. The signal molecules are shown to include the reactive oxygen species, the redox state of the components of photosynthetic electron transport, in particular plastoquinones, the redox-active molecules of plastid stroma, such as thioredoxin and glutathione, and also the intermediates of tetrapyrrole biosynthesis (Mg-protoporphyrin IX and its monomethyl ester). The sophisticated regulatory network is emphasized as a channel matching up the expression of nuclear and plastid genes. The plastid-generated signals help plants adapt to the changing and frequently adverse environmental conditions.     

Upstream stimulatory factor‐2 mediates quercetin‐induced suppression of PAI‐1 gene expression in human endothelial cells

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor‐1 (PAI‐1) in cultured endothelial cells (ECs). Transfection of PAI‐1 promoter‐luciferase reporter deletion constructs identified a 251‐bp fragment (nucleotides ?800 to ?549) responsive to Quer. Two E‐box motifs (CACGTG), at map positions ?691 (E‐box1) and ?575 (E‐box2), are platforms for occupancy by several members of the c‐MYC family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)‐1 and USF‐2 as E‐box1/E‐box2 binding factors. ECs co‐transfected with a 251 bp PAI‐1 promoter fragment containing the two E‐box motifs (p251/luc) and a USF‐2 expression vector (pUSF‐2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF‐2 decreased, while transfection of a dominant‐negative USF construct increased, EC growth consistent with the known anti‐proliferative properties of USF proteins. Quer‐induced decreases in PAI‐1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols. J. Cell. Biochem. 111: 720–726, 2010. © 2010 Wiley‐Liss, Inc.     

Singlet oxygen‐triggered chloroplast‐to‐nucleus retrograde signalling pathways: An emerging perspective

Singlet oxygen (¹O₂) is a prime cause of photo‐damage of the photosynthetic apparatus. The chlorophyll molecules in the photosystem II reaction center and in the light‐harvesting antenna complex are major sources of ¹O₂ generation. It has been thought that the generation of ¹O₂ mainly takes place in the appressed regions of the thylakoid membranes, namely, the grana core, where most of the active photosystem II complexes are localized. Apart from being a toxic molecule, new evidence suggests that ¹O₂ significantly contributes to chloroplast‐to‐nucleus retrograde signalling that primes acclimation and cell death responses. Interestingly, recent studies reveal that chloroplasts operate two distinct ¹O₂‐triggered retrograde signalling pathways in which β‐carotene and a nuclear‐encoded chloroplast protein EXECUTER1 play essential roles as signalling mediators. The coexistence of these mediators raises several questions: their crosstalk, source(s) of ¹O₂, downstream signalling components, and the perception and reaction mechanism of these mediators towards ¹O₂. In this review, we mainly discuss the molecular genetic basis of the mode of action of these two putative ¹O₂ sensors and their corresponding retrograde signalling pathways. In addition, we also propose the possible existence of an alternative source of ¹O₂, which is spatially and functionally separated from the grana core.     

Regulation of endosomal clathrin and retromer‐mediated endosome to Golgi retrograde transport by the J‐domain protein RME‐8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG‐14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome‐associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J‐domain protein RME‐8 associates with the retromer component SNX‐1. Loss of SNX‐1, RME‐8, or the clathrin chaperone Hsc70/HSP‐1 leads to over‐accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG‐14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME‐8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.     

Experimental reconstruction of double‐stranded break repair‐mediated plastid DNA insertion into the tobacco nucleus

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double‐stranded DNA breaks into the nuclear genome of tobacco through inducible expression of I‐SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double‐stranded DNA break repair. Microhomology‐mediated linking of disparate segments of chloroplast DNA occurs frequently during healing of induced nuclear double‐stranded breaks (DSB) but the resulting nuclear integrants are often immediately unstable. Non‐Mendelian inheritance of a selectable marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the screen. The instability of these de novo nu clear insertions of p last id DNA (nupts) was shown to be associated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of transfer. This deletion of pre‐existing nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is potentially far greater than previously thought.