Plant Organelles-to-Nucleus Retrograde Signaling

Plants, unlike animals, are sessile. This demands that adverse changes in their environment are quickly recognized, distinguished and responded to with suitable reactions. Drought, heat, cold and salinity are among the major abiotic stresses that adversely affect plant growth and productivity. In general, abiotic stress often causes a series of morphological, physiological, biochemical and molecular changes that unfavorably affect plant growth, development and productivity. Drought, salinity, extreme temperatures (cold and heat) and oxidative stress are often interrelated; these conditions singularly or in combination induce cellular damage. To cope with abiotic stresses, of paramount significance is to understand plant responses to abiotic stresses that disturb the homeostatic equilibrium at cellular and molecular level in order to identify a common mechanism for multiple stress tolerance. This multi authored edited compilation attempts to put forth an all-inclusive biochemical and molecular picture in a systems approach wherein mechanism and adaptation aspects of abiotic stress are dealt with. The chief objective of the book hence is to deliver state of the art information for comprehending the effects of abiotic stress in plants at the cellular level


Introduction
Plant cells contain two types of energy producing organelles: chloroplasts, which convert solar into chemical energy, and mitochondria, which convert stored energy into ATP.Cell organelles comprise thousands of various proteins; most of them are encoded by nuclear genes; only few genes, which encode mainly the components of the system of gene expression per se and of the respiratory (in mitochondria) or photosynthetic (in chloroplasts) chains, are localized in the genomes of organelles.Thus, the maintenance of cell organelle functional activity during cell growth and development depends predominantly on the nuclear genome encoding most organelle proteins and their own genomes encoding the limited but substantial number of proteins.In this connection, organelle ribosomes, photosystems, complexes of mitochondrial respiratory chain are mosaic in their origin; they are multiprotein complexes comprising subunits encoded by the nucleus and organelles.Therefore, special mechanisms are required for coordination of gene expression in various cell compartments.Since the nuclear genome plays a key role in the biogenesis and functioning of cell organelles, the main recent attention was paid to the analysis of so called anterograde regulation, which controls the flow of information from the nucleus and cytoplasm to organelles.Along with anterograde regulation, retrograde signaling occurs in the cells, when signals emitted by organelles control gene expression in the nucleus (Pesaresi et al. 2007;Yurina & Odintsova, 2007).In this review, we summarize the recent understanding of chloroplast-to-nuclear and mitochondria-to-nuclear retrograde regulation in plant, which involves multiple potential signaling pathways in relation to abiotic stress.

Plastid-generated signals and their role in nuclear gene expression
The very first evidence for the existence of plastid-generated signals that control the expression of nuclear genes encoding the chloroplast proteins was obtained about 30 years ago in the studies of albostrians and Saskatoon mutants of barley (Hordeum vulgare L., cv.Haisa) (Emanuel et al., 2004).The subsequent studies of mutant plants with impaired carotenoid biosynthesis (manifested as photobleached plastids) demonstrated that the absence of functionally active chloroplasts considerably diminished the expression of several photosynthetic genes residing in the nucleus (Oelmüller et al., 1986).Similar results were obtained when carotenoid biosynthesis in the seedlings was reduced by norflurazon, an inhibitor of phytoene desaturase, the enzyme participating in carotenoid biosynthesis (Yurina et al., 2006).In addition to photobleaching, the expression of nuclear genes was inhibited when chloroplast development was blocked by inhibiting the expression of plastid genes with tagetitoxin or nalidixic acid (Gray et al., 1995).Numerous signaling pathways were found to function in the cell and coordinate the expression of nuclear genes depending on chloroplast requirements (Beck, 2005).Protein synthesis in plastids was found to correlate with the expression of nuclear genes coding for plastid proteins.The contents of several nuclear-encoded proteins transferred to plastids, e.g., the Calvin cycle enzymes, and also of proteins functionally related to plastids, such as nitrate reductase, and to peroxisomes, such as glycolate oxidase, catalase, and hydroxypyruvate reductase, were reportedly lower in the leaves of albostrians barley with deficient chloroplast ribosomes than in the wild-type plants.The activity of nuclear genes encoding the chloroplast-localized enzymes in the seedlings of white mustard (Sinapis alba L.) declined in the presence of chloramphenicol, an inhibitor of translation in chloroplasts, whereas the activity of phytochrome-induced cytoplasmic enzymes, such as chalcone synthase, was not affected and in some cases even increased (Oelmüller et al., 1986).In pea (Pisum sativum L.) plants, chloramphenicol inhibited the red-and blue light-induced expression of the gene encoding early light-induced protein (ELIP) (Adamska, 1995).Treating plants with specific inhibitors of translation in chloroplasts, such as lincomycin, erythromycin, and streptomycin, also down-regulated the expression of nuclear genes encoding the photosynthetic proteins.The inhibitors of protein synthesis were shown to affect the expression of nuclear genes only at the early stages of seedling development, during the initial two-three days of germination, and the authors presumed that the products of protein synthesis at the early steps of germination may act as plastid signals (Gray, 1995).As a whole, the evidence already amassed presumes that protein synthesis in plastids generates some signal forerunning the expression of several nuclear genes.These nuclear genes encode the plastid components and also the proteins localized in other cell compartments.However, the data are missing as to whether the inhibition of protein synthesis in the plastids would decrease the expression of any nuclear genes.The current experimental evidence lists among the probable sources of chloroplast signals the reactive oxygen species (ROS), the changes in the redox state of the components of photosynthetic electron transport chain (ETC) of the stromal components of plastids, and also the metabolites produced in the course of photosynthesis (Piippo et al., 2006).In plants, ROS are continuously synthesized as byproducts of numerous metabolic pathways in various cell compartments.ROS include oxygen ions, free radicals, and inorganic and organic peroxides.The contents of ROS are dramatically elevated under stress conditions, such as high illuminance, low temperature, etc. ROS accumulation provokes oxidative stress and damages cell structures.ROS are neutralized by enzymes, such as superoxide dismutase and catalase, and by antioxidant systems.The singlet oxygen 1 O 2 produced by PS II and the superoxide anion generated by PS I are the major forms of ROS produced by chloroplasts under the stress conditions of high illuminance; the superoxide anion is rapidly dismutated into hydrogen peroxide (Apel & Hirt, 2004).Both these ROS were shown to act as plastid signals.The singlet oxygen 1 O 2 generated in plastids affects the expression of several nuclear genes.Plastids of the flu mutant of Arabidopsis thaliana amass protochlorophyllide (protoChlide), which generates the singlet oxygen under illumination.In Arabidopsis, the singlet oxygen was shown to enhance the expression of 70 genes and 57 inhibit the expression of nine genes.Two arguments are put forward to advance the idea that the singlet oxygen serves as a chloroplast signal.First, it is short-lived, about 200 ns, with the transduction distance (the action at the distance) up to 10 nm.Therefore it is thought to perform the role of a plastid-generated signal, which specifically activates the genetically determined program of cell responses to the stress conditions.The EXECUTER 1 protein recently identified in Arabidopsis plants is presumed to recognize or transduce this signal.The N-terminal region of this protein resembles the signal sequences of chloroplast proteins encoded by nuclear genes and essential for the import of these proteins (Wagner et al., 2004).Localization of EXECUTER 1 in plastids would establish an important segment of the signaling pathway linking the plastid-generated singlet oxygen to the expression of nuclear genes.The singlet oxygen is the major constituent of ROS generated by PS II in plants that are deficient in carotenoids due to mutations or following the treatment with herbicides, which inhibit carotenoid synthesis, such as norflurazon.In carotenoid-deficient plants, the plastid structure is considerably damaged, and the expression of several nuclear genes, mostly encoding the photosynthetic proteins, is inhibited.However, the expression profiles of the nuclear genes inhibited by carotenoid deficiency differs from those activated or inhibited by the singlet oxygen generated by illumination in the protoChlide -accumulating flu mutants.Therefore the singlet oxygen generated by PS II in the absence of carotenoids most probably activates the signaling pathway different from the pathway activated by the singlet oxygen produced from protoChlide or generated by PS II in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (Wagner et al., 2004).The idea of alternative signaling pathway was substantiated by the analysis of gun mutants with lower sensitivity of gene expression than in the wild-type plants (Beck, 2005).Hydrogen peroxide is another form of ROS generated by chloroplasts.It accumulates when plants are moved from temperate to strong light, and the superoxide radicals generated by PS I are transformed into hydrogen peroxide.The latter is reduced to water by chloroplast ascorbate peroxidase (APX).While the singlet oxygen, due to its high reaction capacity, mostly remains in the chloroplasts, the excess hydrogen peroxide easily diffuses across the chloroplast envelope into the cytoplasm.Treating plants with H 2 O 2 was shown to induce the expression of nuclear genes related to plant responses to stress factors; thus, the expression of cAPX , the gene for cytoplasmic APX, is enhanced by excess light.The crucial role of hydrogen peroxide in cAPX expression was demonstrated in the experiments with Arabidopsis leaves infiltrated with catalase before excess light treatment: while the induction of APX2 (Arabidopsis cAPX) was inhibited, the expression of the superoxide dismutase gene was not affected (Chang et al., 2004).Recent studies of transgenic tobacco plants expressing the genes for catalase or thylakoidtype APX demonstrated that APX expression involved both hydrogen peroxide and the redox state of plastoquinone pool.Under excess light stress, cell levels of H 2 O 2 in the transgenic tobacco plants were considerably lower and the induction of cAPX was saturated much earlier than in the wild-type plants.It follows that chloroplast-generated H 2 O 2 would represent a redox signal to activate cAPX expression in the nucleus.The initial induction observed under photooxidative stress conditions was related to the redox state of plastoquinone pool (see below for more details), whereas the subsequent mRNA accumulation was in line with continuous accumulation of H 2 O 2 (Yabuta et al., 2004).The correlation between the APX2 expression and H 2 O 2 generation under the excess light stress was also elucidated by comparing the expression of luciferase gene under the APX2 promoter to H 2 O 2 accumulation demonstrated by cytochemical staining with methylviologen (Chang et al., 2004).This dye was shown to enhance the formation of singlet oxygen and hydrogen peroxide in chloroplasts, and the concomitant cAPX expression correlated with the methylviologen-generated H 2 O 2 accumulation in the cells.Both the location and the mechanism of action of plastid-generated H 2 O 2 are unknown.Similar to water, hydrogen peroxide is thought to freely diffuse across the biological membranes and in this way directly interact with out-of-plastid systems of signal transduction.Nonetheless it is not clear how cells discern between the plastid-generated H 2 O 2 and hydrogen peroxide produced in other cell compartments, e.g., on the plasma membrane when cells are attacked by pathogens.The stress responses to pathogens are known to widely differ from those induced by excess light stress (Beck, 2005).Thus, we conclude that at least two chloroplast-generated forms of ROS, the singlet oxygen and hydrogen peroxide, participate in the transduction of specific signals from the plastids to the nucleus.Two ROS forms induce different responses at the level of gene expression and probably perform in the different signaling pathways.The mechanism of ROS interaction with the nucleus is also unknown.In this aspect, the experiments with mutants, such as EXECUTER1 where the singlet oxygen participates in the plastid signal blockade, are very promising (Beck, 2005).The environmentally induced changes in redox state of the components of photosynthetic ETC act as signals that regulate gene expression in the chloroplasts and partly in the nucleus (Pfannschmidt & Liere, 2005).It means that photosynthesis is a source of information essential for the control over the nuclear gene expression that is not recognized by cytoplasmic photoreceptors, while the chloroplasts themselves serve as sensors for the changes in light quantity and quality and in this way induce the physiological responses of photoacclimation (Beck, 2005).The participation of the redox state of ETC components in nuclear gene expression was established in several ways.To change the redox state of ETC components, plants grown at low light were transferred at regular intervals into strong light and back.The same goal was attained by the light conditions that primarily excited either PS I or PS II, by changing the growth temperature and the level of carbohydrates and electron acceptors, such as O 2 and CO 2 , and by herbicides DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which specifically blocked electron transport from PS II to the cytochrome b 6 /f complex at the site upstream (DCMU) or downstream of plastoquinone pool (DBMIB).These data led to the conclusion that the redox state of plastoquinones was the initial signal that regulated the expression of particular genes (Nott et al., 2006).Recently the role of the redox state of ETC as the source of chloroplast signal(s) transferred into the nuclear compartment has been supported by the analysis of expression profile of numerous nuclear genes in Arabidopsis: 2661 out of 3292 genes under analysis encoded chloroplast proteins and only 631, nonchloroplast ones (Richly et al., 2003).In the cases when the plants grown under PS I-inducing light were transferred under PS II-inducing illumination, the changes in mRNA contents were registered for 2133 genes: the changes were positive (enhanced expression) for 1121 genes and negative (diminished expression) for 1012 genes.The in depth study demonstrated that among these genes, 286 are immediately regulated by the redox state of photosynthetic ETC signals: the expression of 86 genes was up-regulated and 200 down-regulated (Fey et al., 2005).Whatever small were the changes in the expression levels of most genes, this evidence seems to prove that the redox state of the ETC components regulate the expression of many nuclear genes.In green algae, the redox state of the plastoquinone pool was shown to affect Lhcb expression.In higher plants, however, expression of Lhcb has been correlated with the phosphorylation status of LhcII.The thylakoid protein STN7 has been identified as the protein kinase for LHCII (Inaba, 2010).The control exerted by the redox state of plastoquinone pool on the nuclear gene expression in the higher plants is of lesser importance than in the green algae.Chloroplast redox signals were shown to affect the nuclear gene expression at both transcriptional and posttranscriptional (Fed-1, PetE) levels by controlling mRNA stability and binding to polyribosomes (Sherameti et al., 2002).The redox state depends on illumination, and therefore the photosynthetic apparatus can be seen as a photoreceptor recording photon quality and quantity (Beck, 2005).In addition to the redox state of chloroplast ETC components, the nuclear genes are also controlled by the redox state of the stromal components, such as glutathione and the ferredoxin-thioredoxin system.It follows that in mature leaves of higher plants, the redox state of the components at the donor side of PS I is more important for light-dependent changes in the expression of nuclear genes coding for plastid proteins than the redox state of plastoquinone pool, especially at the early steps of signal transduction.Such conclusion confers with the evidence from the experiments with Synechocystis sp.6803: in the latter case, the expression of few genes was related to the redox state of plastoquinone pool (Piippo et al., 2006).Following short exposure to extreme environmental conditions, the leading role in signal transduction from the chloroplasts to the nucleus would pass on to the stromal redox components, the products of CO 2 fixation, and the ATP/ADP ratio.Recently many studies were focused on the role of Mg-protoporphyrin IX (MgProtoIX) and its methyl ester (MgProtoIXMe) as the regulators of the nuclear gene expression.The first proof for the participation of chlorophyll precursors in the transduction of plastid signals was obtained in the experiments with Chlamydomonas reinhardtii cells.In synchronous cultures of this alga, mRNA of the gene Lhcb accumulated already in two hours after the cultures were transferred into the light, mostly due to enhanced transcription.The studies of higher plants support the idea that chlorophyll precursors participated in the transduction of plastid-generated signals.Thujaplicin treatment of etiolated cress seedlings interfered with protochlorophyllide synthesis and resulted in MgProtoIX accumulation and decline in the light-induced synthesis of Lhcb mRNA.When treated with 5-aminolevulinic acid (ALA), such seedlings produced only half of light-induced mRNA Lhcb as compared to the seedlings treated with water.When treated with amitrole, an inhibitor of carotenoid biosynthesis, the ethiolated barley seedlings accumulated ALA, MgProtoIX, and MgProtoIXMe, with the concomitant decline in light-induced Lhcb and RbcS expression.In Arabidopsis, several nuclear genes encode proteins essential for plastid-to-nucleus signal transduction have been identified (Susek et al., 1993).The Lhcb promoter equipped with the proper selection marker was built into the nuclear genome, and seeds were mutagenized with ethyl methanesulfonate.These lines were used to isolate mutants with Lhcb expression independent of the chloroplast functional state.While due to chloroplast photobleaching, the expression of reporter genes under the Lhcb promoter was low in the wild-type plants grown under continuous illumination in the presence of norflurazon, the mutants exhibited high expression of Lhcb on the norflurazon-supplemented medium.Such screening identified five nonallelic loci with diminished capacity for plastid-to-nucleus signaling; these mutants were called "genomes uncoupled", or gun-mutants (Susek et al., 1993).Four gun-mutants have been already characterized at the molecular level: several impaired enzymes, such as heme oxygenases, phytochromobiline synthases, and H subunits of Mgchelatase or MgProtoIX-binding protein, were localized in the plastids and shown to participate in the porphyrin biosynthesis.The mutations at these genes decreased MgProtoIX accumulation.Damaging other enzymes in the biosynthetic pathway leading to MgProtoIX also diminished the plastid control of Lhcb expression.The plastid signal transduction is apparently affected by plant developmental stage: while the expression of Lhcb, HEMA1, and Elip was inhibited in norflurazon-treated seedlings of Arabidopsis and barley, similar treatment of adult plants downregulated only Lhcb expression (Pogulskaya et al., 2006).A considerable progress has been recently reported in characterizing the sequences of the target genes for plastid signals, such as MgProtoIX and others.The study of light-responsive promoters of the genes controlled by plastid signals and mostly encoding the components of photosynthetic apparatus demonstrated that light and plastid signals affected the nuclear gene expression at one and the same cis-elements.In these experiments (Kusnetsov et al., 1996), the plastid signal controlling the nuclear gene expression was activated with norflurazon, which promoted photooxidation of the thylakoid membrane in the light.Based on the above-mentioned data, it was suggested that in higher plants a plastid-tonucleus signal is induced by MgProtoIX and/or MgProtoIXMe (Nott et al., 2006;Pogulskaya et al., 2006;Strand et al., 2003;Yurina et al., 2006).Subsequent studies showed that accumulation of MgProtoIX is not always accompanied by inhibition of Lhcb expression (Mochizuki et al., 2008;Mochizuki et al., 2010;Moulin et al., 2008;).Determination of ProtoIX, MgProtoIX and MgProtoIXMe concentrations in Arabidopsis seedlings grown in the presence of norflurazon showed that norflurazon inhibits significantly the formation of the intermediates of tetrapyrrole biosynthesis, the inhibition was more pronounced in older seedlings.The expression levels of some other genes, such as Lhcb1, RbcS, HEMA, BAM3 (encodes beta-amylase) and CA1 (encodes carbonic anhydrase) were also reduced.It was also shown that the Arabidopsis mutants cs and ch42 with impaired Mg-chelatase subunit I (ChII) did not display gun phenotype, although the production of MgProtoIX in these mutants was considerably decreased (Mochizuki et al., 2001).The herbicide 2,2'-dipyridyl supposed to induce accumulation of the MgProtoIXMe inhibited gun2 and gun5 phenotypes (Mochizuki et al., 2008).In norflurazon-treated seedlings, the Lhcb expresssion was decreased, however no accumulation of porphyrins was observed (Gadjieva et al., 2005).Contradictory data concerning the signaling role of MgProtoIX in the repression of nuclear plastid protein genes may be associated with problems of accurate quantitative determination of tetrapyrrole biosynthesis intermediates.A detailed study of the role of MgProtoIX (and other chlorophyll biosynthesis intermediates) as signaling molecules during retrograde regulation revealed the absence of correlation between MgProtoIX accumulation and expression of nuclear plastid protein genes.It was shown that in norflurazon-treated plants at different growth conditions, nuclear gene expression was inhibited, however, the accumulation of MgProtoIX or other intermediates of chlorophyll biosynthesis did not occur (Moulin et al., 2008).Conversely, elevation of the MgProtoIX endogenous level by the addition of the tetrapyrrole precursor ALA caused an induction not repression of the nuclear photosynthetic genes.Chemical or genetic modification of the tetrapyrrole levels and light conditions revealed no correlation between the intermediates of tetrapyrrole biosynthesis (including MgProtoIX) and Lhcb expression (Mochizuki et al., 2010).Gun mutations had no effect on heme accumulation (Voigt et al., 2010).The determination of tetrapyrrole content in Arabidopsis plants under light stress conditions by a conventional procedure revealed no significant changes in MgProtoIX concentration in norflurazon-treated plants as compared to control plants (our data).A certain decrease in MgProtoIX concentration was even recorded (unpublished data).Thus, it remains unclear whether MgProtoIX serves as a signal during retrograde regulation in higher plants.Based on conflicting experimental data it was suggested that not MgProtoIX molecules but their derivatives, degradation p r o d u c t s o r R O S , s u c h a s singlet oxygen or hydrogen peroxide, may induce retrograde signaling cascades (Mochizuki et al., 2010;Moulin et al., 2008).There is experimental evidence that expression of nuclear plastid protein genes Lhcb and RbcS is controlled by hydrogen peroxide and singlet oxygen (La Rocca et al., 2001).
Based on these data it was hypothesized that a simple signaling cascade mechanism connecting MgProtoIX accumulation with inhibition of nuclear plastid protein gene expression is unlikely (Mochizuki et al., 2008;Moulin et al., 2008).The discrepant results may partially arise from different conditions of norflurazon treatment of seedlings (Zhang et al., 2011).It cannot be excluded that MgProtoIX-generated signals are short-living and function in particular cellular compartments and thus are difficult to detect.Transcriptome analysis of the green unicellular algae C. reinhardtii grown in the presence of MgProtoIX or heme showed that tetrapyrrole biosynthesis intermediates transiently but considerably changed expression of approx.1000 genes.They include a limited number of photosynthesis-related genes, the genes for the tricarbonic acid cycle enzymes, the genes for heme-binding proteins, several stress-responsive genes, as well as genes involved in protein folding and degradation.Both tetrapyrroles act as secondary messengers in adaptive response of the whole cell, not only cellular organelles (Voss et al., 2011).Another important process that may generate a plastid signal is protein import into plastids (Inaba, 2010).It was shown that in a mutant with a deficiency of the main receptor for the imported proteins Toc159, expression of nuclear plastid protein genes is inhibited.This abnormality in protein import into the plastids serves as a plastid signal (Kakizaki & Inaba, 2010).By this mechanism retrograde signals regulate expression of nuclear plastid protein genes in accordance with the requirements of these organelles and provide efficient assembly of multisubunit complexes encoded both by nuclear and chloroplast genes.It should be noted that the signaling molecules triggering retrograde regulation have not been identified so far (Pfannschmidt, 2010).

Components of the retrograde signaling pathways
The GUN1 protein plays an important role in retrograde signal transduction.It was shown that the gun1 mutation is not associated with tetrapyrrole biosynthesis.This follows from the differences between the gun1 and gun2-gun5 genes (Cottage et al., 2010).Treatment of plastids with lincomycin, an inhibitor of protein synthesis, represses expression of nuclear photosynthetic genes in wild-type Arabidopsis seedlings and gun2-gun5 mutants, but not in gun1 mutants (Koussevitzky et al., 2007).The gun1 gene codes for a protein of plastid nucleoids, which contains 10 copies of a pentatricopeptide repeat (PPR) and the so-called SMR-domain (a minor MutS-associated domain) close to the C-terminus of the polypeptide chain (Cottage et al., 2010;Koussevitzky et al., 2007).The SMR-domain binds DNA, while the PPR-motif participates in RNA processing and is found in many mitochondrial and plastid proteins.Since lincomycin does not inhibit expression of nuclear photosynthesisrelated genes in gun1 mutants, it was suggested that GUN1 may be involved in signal transduction induced by impaired expression of plastid genes (Armbruster et al., 2011).GUN1-dependent signal transduction may also be involved in coordination of nuclear photosynthetic gene expression with the efficiency of protein import into the plastids (Kakizaki & Inaba, 2010).It was demonstrated that GUN1 mediates signals induced by tetrapyrrole biosynthesis intermediates and redox state of the electron transport chain.Since gun1 mutants display abnormal reaction to high-intensity light, it was hypothesized that GUN1 integrates signals induced by norflurazon, lyncomycin and high-intensity light (Koussevitzky et al., 2007).A comparative study of anthocyan and Lhcb1 transcript accumulation in gun1-1 mutants and wild-type Arabidopsis seedlings indicated that the deficiency of functional GUN1 impairs early development of seedlings and alters the sensitivity of plants to sucrose and abscisic acid (see below).However, the mode of GUN1 action is presently unknown (Cottage et al., 2010).In the retrograde signaling pathway downstream of GUN1 operate two nuclear transcription factors: AP2 (Apetala 2)-like transcription factor ABI4 and GLK1.The Arabidopsis abi4 mutant has a weak gun phenotype.The abi4 gene is highly expressed in seeds in contrast to seedlings.The abi4 expression is induced by glucose and probably other sugars.ABI4 is a negative regulator of the Lhcb expression.In response to plastid signals ABI4 competitively binds to G-box of the cis-element and inhibits Lhcb expression (Inaba, 2010).The transcription factors of the Apetala 2 (AP2)-type act as repressors of transcription in the presence of abscisic acid, ethylene and jasmonic acid (Koussevitzky et al., 2007).By a still unknown mechanism GUN1 activates ABI4.It was shown that just this mechanism is used by retrograde signals induced by tetrapyrrole biosynthesis intermediates, plastid gene expression and redox state of the electron transport chain, but differs from the mechanisms utilized by signals associated with abnormal protein import into the plastids (Inaba, 2010).In this case, the transcription factor GLK1 operates.In contrast to ABI4, GLK1 is a positive regulator of Lhcb expression, which coordinates expression of nuclear photosynthetic genes (Kamikaze & Inaba, 2010).Under stressful conditions, such as norflurazon treatment and impaired protein import, GLK1 expression is considerably decreased that leads to inhibition of photosynthesis-related genes.For GLK1 repression, GUN1 is necessary.It was shown that many nuclear photosynthetic genes controlled by plastid signals contain the ACGT sequence in their promoter regions that also serves as the major (core) element involved in cell response to abscisic acid.This points to the involvement of components of ABA-signaling cascade (or abscisic acid levels) in retrograde signaling (Jung & Chory, 2010;Koussevitzky et al., 2007).Analysis of the ABA-deficient (aba) and ABA-insensitive (abi) Arabidopsis mutants showed that in regulation of nuclear gene expression and plant adaptation to stressful conditions, the components of the ABA cascade are tightly associated with the Mg-Proto retrograde signaling pathway.The interaction between the plastid and abscisic acid signals is mediated by ABI4, which is a 'master switch' that controls expression of a large number of genes in response to diverse signals.The ABI4 transcription factor is not only a component of the plastid retrograde signaling pathway but is also involved in mitochondrial retrograde signaling, thus it serves as a convergence point of these signaling pathways (Cottage et al., 2010).
It was shown that the H subunit of Mg-chelatase (ChIH) participates in signal transduction from the plastids to the nucleus and in ABA-dependent signaling cascades.The ChIH protein is a receptor of ABA-induced signals.In Arabidopsis it is a positive regulator of signaling induced by Mg-Proto and chlorophyll biosynthesis intermediates.Plants with enhanced expression of this protein are supersensitive to abscisic acid, while ChIH-deficient mutants possess an ABA-insensitive phenotype.It was assumed that the plastid proteins EX1 and EX2 (Inaba, 2010) and protein kinases, in particular the thylakoid membrane protein STN7, which is a Lhcb protein kinase (Inaba, 2010;Pfannschmidt, 2010), are also involved in retrograde signaling.However these proteins are located in the plastids.The cytosolic mediators of the plastid signals have not been identified.The nuclear transcription factor Whirly1 (Why1), which was originally described as a telomere-binding protein, is also supposed to be involved in retrograde signaling.Abundant experimental data indicate that plastid retrograde signaling cascades interact with light signaling.Light activates expression of a large number of photosynthesis-related nuclear genes, such as Cab encoding chlorophyll a/b -binding proteins, RbcS (encoding the small subunit of Rubisco) and Pc coding for plastocyanin.Retrograde signals may act synergistically with the light signals or transform them from inducers into repressors of photosynthetic gene expression (Larkin et al., 2008;Osipenkova et al., 2010).It is generally accepted that the plastid retrograde signals are endogenous regulators of the light signaling and that integration of the light and plastid signals helps plants to overcome chloroplast dysfunction during organelle biogenesis under unfavorable light conditions (Larkin et al., 2008).Both types of signals act on the same cis-elements in the promoters of the nuclear plastid protein genes (Kusnetsov et al., 1996).The ABI4 participates both in retrograde and light signaling.This protein binds to the promoter region of the gene regulated by a retrograde signal through the conserved CCAC motif, which is a core element necessary for binding.In such a way ABI4 inhibits G-box mediated light-inducible expression of photosynthesis-related genes when the chloroplast development is inhibited (Koussevitzky et al., 2007).Earlier a strong correlation between the light and sucrose signaling pathways was revealed (Dijkwel et al., 1997).It was shown that the sucrose signaling pathways may interact (at least in some tissues) with other retrograde signaling pathways and thus modulate the response of nuclear genes to retrograde signals.In the study of sun6 (sucrose-uncoupled6) Arabidopsis mutants (allelic abi4-mutants), a correlation between redox signals and sucrose-regulated gene expression was established (Oswald et al., 2001).It was also shown that the SUN6 protein is involved in sucrose repression of phytochrome A-dependent signaling pathways (Dijkwel et al., 1997).These data indicate interactions of sucrose, light and plastid signaling pathways.The data presented indicate that plant cells possess a complex network of signaling pathways that function independently or interact with each other due to common intermediates or final cascade components.Tetrapyrroles play a key role in this network, their metabolism regulates the plant cell functions.The transfer of numerous genes from cell organelles into the nucleus in the course of cell evolution (Odintsova & Yurina, 2006) led to gene redistribution in cell compartments and the coordinated expression of nuclear and organellar genes.The expression of nuclear genes i n p l a s t i d s a n d m i t o c h o n d r i a m u s t b e h a r m onized with the functional state of these organelles, and such coordination was provided by evolution of the so-called retrograde (organelle-to-nucleus) control over the expression of nucleus-coded organellar genes.The pathways of such signal transduction have not been sufficiently clarified: we do not know how the plastid or mitochondrial signal crosses the organelle membranes, what signal molecules stay in the cytosol and nucleus, and whether their action upon the regulatory proteins of the nucleus is direct or mediated by the induction of other signals.In most cases the plastid signals are related to the redox state of organelles, e.g., the ETC components and redox-active stromal compounds, such as thioredoxin and glutathione, or with the chlorophyll biosynthesis.ROS, the side products of photosynthesis and mitochondrial respiration, also can participate as signal molecules in the organelle-to-nucleus signal transduction.ROS are formed by both types of organelles, and therefore can provide for the coordinated expression of nuclear genes in the plastids and mitochondria at the transcriptional level.The analysis of the nucleus-encoded chloroplast transcriptome demonstrated that the transcription of the nuclear genes coding for plastid proteins was controlled by several types of plastid signals comprising the complicated signal network within plant cells (Leister, 2005).The attempts to investigate this network using the mutant plants are problematical because alternative control mechanisms may function in the mutants impaired in one and the same photosynthetic complex (Beck, 2005).

Mitochondrial retrograde regulation in plants
Most data about mitochondrial retrograde regulation (MRR) in plants were obtained on material with disturbed functioning of mitochondria.Expression of some nuclear genes is activated in response to disturbances in mtETC, tricarbonic acid cycle, and also mtDNA (Rhoads & Subbaiah, 2007).Plant mitochondria dysfunction induced by mutations results frequently in male sterility, embryo lethal phenotype, or chlorosis in plants, which could not complete their life cycle (Newton et al., 2004).Plant mitochondria dysfunction can be induced by biotic and abiotic stresses.A bulk of information was obtained demonstrating a great contribution of mitochondria in general plant response to stress.Mitochondrial retrograde regulation in plants is usually studied on the systems with disturbed mitochondrial functions induced by mutations, chemical agents, or biotic and abiotic stresses.Dysfunction of mitochondria leads to changes in the nuclear gene expression.Plant response to mitochondrial function disturbance is an expression induction of genes encoding proteins involved in the restoration of mitochondria functioning, such as alternative oxidase (AO) or alternative NADPH-dehydrogenases, and genes encoding antioxidant enzymes, such as glutathione transferases, catalases, ascorbate peroxidases, and superoxide dismutases, normalizing ROS level (Rhoads & Subbaiah, 2007).At present, most studied plant response to mitochondria dysfunction is the changes in expression of the nuclear gene encoding AO (Mackenzie & McIntosh, 1999;Rhoads & Vanlerberghe, 2004).It is believed that the two pathways of signal transduction to the nucleus operate at AO expression: one with the involvement of ROS and another with the involvement of organic acids (Gray et al., 2004).Cytoplasmic male sterility (CMS), i.e., plant failure to produce viable pollen, is induced by signals from mitochondria and is one of the characteristic examples of MRR in plants.Male sterility is often results from mutation-induced mitochondria dysfunction.Changes in mtDNA or mitochondrial gene expression induce changes in nuclear gene expression, result in the modification of stamen phenotype, and finally in the suppression of pollen formation.CMS is a commonly occurring phenomenon observed in more than 150 plant species.It is met frequently in hybrid lines obtained due to intra-or inter-specific crosses, i.e., in alloplasmic lines where the nucleus of one species functions in the cytoplasm of another species.The fact that CMS is determined by the interaction between the nucleus and mitochondria was proved by maternal inheritance of male sterility phenotype and by suppression of male sterility with nuclear genes of fertile restoration (Rf genes).These genes (except a single gene) encode proteins belonging to the family of PPR proteins (proteins containing pentatricopeptide repeats).These proteins in Arabidopsis are supposed to be localized in mitochondria or plastids.Some of them participate in the processing of various RNAs in organelles (Carlsson et al., 2008).It should be noted that not only retrograde signaling pathways but also other mechanisms could result in CMS, i.e., diverse mechanisms could determine male sterility.However, the target genes of retrograde signaling pathways and functions of genes for fertility restoration are evidently common for many CMS systems.Since several Rf genes could suppress the phenotype of male sterility and restore male fertility in various species, we can suppose that changes in transcription, translation, or RNA processing in mitochondria are represent the common mechanism of restoration.The homeotic genes, i.e., genes, which mutations result in changes of floral organs, are most probable targets for the retrograde signaling pathways directed from CMS-inducing mitochondria to the nucleus.However, the signal for CMS origin is unknown (Carlsson et al., 2008;Kubo & Newton, 2008).Plant mitochondria respond to abiotic and biotic stresses.They can serve stress sensors and initiate responses (or be involved in responses) to particular stress types.Thus, MRR is involved in the plant cell response to oxygen stress.It is known that, under definite conditions (flooding for example), oxygen is a limiting factor for plant growth and survival.Under normal growth and development, plant also could experience hypoxia because oxygen diffusion in tissues with closely packed cells and small intercellular spaces is hindered.It is also known that even plants acclimated to land conditions manifest a great tolerance to oxygen deficit, which indicates the presence in them of highly sensitive system for oxygen sensing and well developed responses to oxygen stress (Bailey-Serres & Chang, 2005).Adaptation to a rapidly changing oxygen level includes rapid changes in gene expression (potentially induced by preceding rapid changes in the level of ROS, redox state, and/or energetic status) permitting a correction of the metabolism (for example, suppression of carbon entry in TCA cycle with parallel increase in the carbon entry into glycolytic and other enzymic pathways) (Rhoads & Subbaiah, 2007).Reactions leading to programmed cell death are also one of the plant responses to anaerobiosis (Subbaiah & Sachs, 2003).The genes controlled by oxygen could be roughly divided into aerobic genes (transcribed at atmospheric oxygen level), hypoxic genes (induced at lowered oxygen level), anoxic genes (induced in the absence of oxygen), and hyperoxic genes (transcribed under oxidative conditions).Certainly, such a division is rather conditional (Rhoads & Subbaiah, 2007).Since oxygen is primarily absorbed by cell mitochondria, changes in the level of available oxygen should be primarily sensed by these organelles, resulting in MRR of nuclear genes.The levels of transcripts and proteins (TCA enzymes or mtETC components) were shown to be reduced at oxygen removal and rapidly restored after oxygen level restoration (Branco-Price et al., 2005).In experiments with maize cell culture, it was shown that, in the absence of oxygen, immediate and reversible increase in the Ca 2+ level in the cytosol ([Ca 2+ ] cyt ) occurs.Changes in the Ca 2+ level are observed around mitochondria and disappeared by the blocker of mitochondrial calcium channel ruthenium red.This dye suppresses induction of genes sensitive to anoxia, alcohol dehydrogenase 1 (adh1) and sucrose synthase (sh1) in maize seedlings and cell culture (Subbaiah et al., 1998).Maize seedlings treated with ruthenium red are especially sensitive to anoxic stress.Ca 2+ addition neutralizes ruthenium red effects, confirming the supposition that ion changes in maize evidently initiated by mitochondria are the signals for responses to anoxia.Calcium ions were shown to be an important component of the signal transduction pathway related to hypoxia in Arabidopsis, rice, and barley, which indicates the conserved nature of this pathway in plants (Rhoads & Subbaiah, 2007).The conclusion that mitochondria are a source of [Ca 2+ ] cyt -signal initiating nuclear gene activation is supported by dynamics of mitochondrial Ca 2+ ([Ca 2+ ] mt ) in response to anoxia.[Ca 2+ ] mt release was shown to occur in maize cells immediately after ceasing oxygen influx.Heat stress is known to reduce crop yield.One of the ways to decline heat shock effects is the induction of genes encoding heat shock proteins (HSPs), including low-molecular HSPs (sHSPs).Trasgenic plants expressing HSPs are a convenient model for studying mitochondrial responses to heat shock.Thus, transgenic Arabidopsis plants were obtained expressing transgene encoding maize mitochondrial sHSP, ZmHSP22 (Rhoads et al., 2007).This protein is weakly constitutively synthesized in etiolated maize seedlings and induced by heat shock.In transgenic line, this transgene under the control of cauliflower mosaic virus 35S promoter was actively expressed constitutively.Using confocal immunofluorescent microscopy and the analysis of isolated mitochondria, it was shown that ZmHSP22 penetrates Arabidopsis mitochondria and processed there with the formation of mature protein.In transgenic plants subjected to heat stress, expression of several nuclear genes encoding endogenous mitochondrial sHSP of Arabidopsis (AtHSP23.6)and also HSPs localized in chloroplasts (AtHSP25.3and AtHSP70-6) and cytosol (AtHSP17.4and AtHSP70-1) was changed.In wild-type plants, AtHSP23.6 is weakly expressed but induced by heat stress.These data allow a supposition that heatinduced MRR could affect HSP expression.The common component of various abiotic stresses is an oxidative stress induced by ROS generation.Oxidative stress could induce mitochondria dysfunction in plants and result in inactivation of definite hormonal signals (Rhoads & Subbaiah, 2007).Proteomic approach for identification of mitochondrial components sensitive to oxidative damage showed that they are enzymes of the TCA cycle, components of mtETC and oxidative phosphorylation (Sweetlove & Foyer, 2004).In the leaves, glycine decarboxylase, the enzyme of the photorespiratory pathway, turned out to be especially prone to oxidative damage (Taylor et al., 2002).

Components of signal transduction pathways related to plant mitochondrial retrograde regulation
The common feature of abiotic and biotic stresses in plants is an increased level of ROS in the cells, which leads to changes in expression of nuclear genes (Vranova et al., 2002).However, the precise role of ROS in plant responses to stresses is unknown.A specific ROS property is that they are damaging compounds and simultaneously signal molecules during plant responses to stresses.However, at different stresses, ROS elevated level induces different changes in gene expression; ROS can interact with other stress-factors, such as calcium, hormones, or changes in the cell redox state (Gadiev et al., 2006).mtROS are the part of the total ROS pool generated in the cells in response to stress.The contribution of mitochondria and other cell compartments into the ROS level is undetermined; changes in nuclear gene expression induced by each of these compartments are unknown as well.It was shown that inhibition of the cytochrome respiratory pathway results in the increase in the mtROS content (especially hydrogen peroxide), which evidently induces MRR signaling and AO gene expression, which leads to the reduction in mtROS generation.However, this does not mean that mtROS are required for all MRR pathways functioning in the plants.Monofluoroacetate is a mighty inducer of AtAOX1a gene expression (Zarkovic et al., 2005), but it does not induce a strong enhancement in ROS generation in the cell (Rhoads & Subbaiah, 2004).Although the role of mtROS and MRR at inhibition of plant mtETC by antimycin seems to be proven, other signaling components of mitochondrial regulation downstream ROS are not identified.It was shown that cell redox state and signals emitted by the photosynthetic ETC are involved in the control of nuclear and chloroplast gene expression.Changes in the redox state have also a great significance for plant responses to stresses (Foyer & Noctor, 2005).Mitochondria can change the cell redox state, in particular due to ROS generation and functioning of the glutathione-ascorbate cycle.It is supposed that in plants glutathione and ascorbate are the components of redox signaling, which induce expression of defense genes (Dutilleul et al., 2003;Foyer & Noctor, 2005).Glutathione is also supposed to be a component of the signal transduction pathway related to cold stress (Kocsy et al., 2001).Until now, only few components of the MRR signaling pathways in plants are identified.Among data available, especially interesting ones are that calcium is involved in MRR related to hypoxia; indeed, Ca 2+ ions participate in signal transduction in mitochondrial retrograde signaling pathways most frequently met in higher eukaryotes (Butow & Avadhani, 2004).It is supposed that Ca 2+ is involved as a signal not only in plant MRR under hypoxia but also in other stress types.All these data argue for an important role of Ca 2+ transport for plant responses to stresses.It is unknown how a signal from the increase in the [Ca 2+ ] cyt determined by mitochondria is amplified, transmitted to the nucleus, and results in changes in nuclear gene expression.It is believed that Ca 2+binding proteins participate in this process, viz., calcium-dependent protein kinases, transcription factors, and regulatory proteins (such as 14-3-3 proteins).It is not excluded that Ca 2+ signal per se is transferred to the nucleus.Some data are obtained that changes in the Ca 2+ content in the nucleus affect directly gene expression.Plant genomes (at least sequenced genomes of Arabidopsis and rice) contain more genes encoding transcription factors than sequenced genomes of other organisms; in particular, they comprise unique gene families characteristic of only plants (Chen et al., 2002).It is supposed that these factors could be additional factors tuning plant responses to stresses.Some of them are evidently involved in MRR, although no transcription factors specific for MRR are identified until now.It might be that such factors as WRKY, bZIP, and Dof are involved in plant MRR because they participate in responses to biotic and abiotic stresses, including oxidative stress (Chen et al., 2002).The elucidation of molecular mechanisms of plant MRR is only at its start.Mitochondria responses to stresses, which are the part of total plant responses, could determine the fate of plant cells, resulting in the restoration of their vital activity or in their death.Thus, induction of AO and alternative NADPH-dehydrogenases in tobacco cell suspension at suppression of cytochrome respiratory pathway prevent cell death.In the cells lacking AO, ROS accumulation induced by mtETC inhibition evidently shifts metabolism toward programmed cell death (Robson & Vanlerberghe, 2002).A capability of the induction of AO expression could also affect the independent mitochondrial pathways of programmed cell death.Several retrograde mitochondrial signaling pathways could function in the plant cells (Zarkovic et al., 2005), and they could initiate specific changes in nuclear gene expression in response to specific disturbances in mitochondria functioning.Retrograde mitochondrial signaling pathways interact with each other.In addition, they interact with retrograde chloroplast signaling pathways (Pesaresi et al., 2006(Pesaresi et al., , 2007) ) and other signal transduction pathways in the plant cell including sugars (Pesaresi et al., 2007), hormones (Kwak et al., 2006), enzymes (Subbaiah et al., 2006), etc.The interaction between retrograde signaling from mitochondria and plastids is of a great interest because these signals control expression of nuclear genes encoding organelle components in dependence on organelle functional state.Chloroplast and mitochondrial metabolisms are known to be connected.Photosynthesis provides substrates for mitochondrial respiration and, in its turn, depends on some compounds synthesized by mitochondria.In darkness, mitochondria are the major source of ATP for cell processes, including those in chloroplasts.In addition in darkness, ATP supports the proton gradient across the thylakoid membrane, thus protecting chloroplasts against photoinhibition after the start of illumination.In the light, mitochondria provide chloroplasts with carboncontaining compounds, produced in the TCA cycle, for NH 4 + assimilation, whereas ATP supports diverse biosynthetic reactions, including the restoration of photosystem II functions (Pesaresi et al., 2006).Data concerning signal molecules and components of inter-organelle signaling pathways are few.The molecular analysis of these pathways supposes their multiple interactions.It is believed that NO, ascorbate, and ROS could fulfill the role of signals in the mitochondria and chloroplast interactions.However, the ways of signal transduction from organelles to the nucleus and the incorporation of these signals into the general system of expression regulation are not known so far (Pesaresi et al., 2006).

Conclusion
Retrograde regulation of nuclear gene expression by organelles is best studied in budding yeast S. cerevisiae and higher plant chloroplasts.In chloroplasts, retrograde signals are mostly related to the redox state of organelles or to chlorophyll biosynthesis.Intermediates of tetrapyrrole biosynthesis and the products of organellar protein synthesis are evidently major retrograde signals (Pesaresi et al., 2006(Pesaresi et al., , 2007)).Changes in the nuclear gene expression dependent on plastid retrograde signaling pathways include a multilevel control of transcription and the involvement of the ABI4 transcription factor (Pesaresi et al., 2007).ROS, harmful by-products of photosynthesis and mitochondrial respiration, could also serve signal molecules transmitting a signal from organelles to the nucleus.Since ROS are generated in both types of organelles, they could provide for coordinated expression of nuclear genes of plastid and mitochondria at the level of transcription.Most identified regulons are shown to contain nuclear genes of chloroplast and mitochondria, and this allows coordinated regulation of activities of these organelles (Rhoads & Subbaiah, 2007).Despite the fact that investigations of plant organelles-to-nucleus retrograde signaling have been the subject of intensive research for several decades, the available data are fragmentary.The molecules, which induce the signal, are still unknown, as well as the mechanisms of signal transduction and the components of the signaling cascade involved.Not much is known on the specificity and cross-talk between different signaling pathways in plants.Usage of classic and novel methodological approaches help identify signal molecules involved in mitochondrial retrograde regulation in plants.Further research exploiting the traditional methods of biochemistry, direct and reverse genetics, proteomics and metabolomics will help to identify the signaling molecules triggering retrograde cascades, to unravel the mechanisms of signal transduction.

Acknowledgment
This study was supported by the Molecular and Cell Biology program of the Presidium of RAS.