However, upon ligation by cognate chemokines, ACRs fail to induce classical signaling and downstream cellular responses characteristic for GPCRs. Despite this, by affecting chemokine availability and function, ACRs impact on a multitude of pathophysiological events and have emerged as important molecular players in health and disease. This review discusses individual characteristics of the currently known ACRs, highlights their similarities and differences and attempts to establish their group identity. It summarizes the progress made in mapping ACR expression, understanding their diverse  Therefore, true subcellular functions of ACRs may be different in vivo and may vary depending on the cellular context.Directed movement of cells defines embryogenesis and importantly contributes throughout life to many physiological and pathological processes. Among different molecules able to provide directional cues and drive augmented-rate cell locomotion, chemokines occupy a unique position in vivo settings for the directionality of cell responses to chemokines. Therefore, even those ACRs, which contribute to chemokine degradation, may support directional chemokine-induced in vivo cell migration. In addition to affecting chemokine positioning within tissues, ACRs may influence the responses to chemokines by heterodimerizing with chemokine GPCRs expressed in cis-geometry, thus modifying their availability and signaling. ACRs may also interfere with or consume secondary intracellular signaling molecules, β-arrestin in particular. Such mechanism characterizes the function of C5L2, an interceptor for chemoattractant C5a Curiously, irrespective of either scavenging or transporting chemokines, ACR activity may lead to the establishment of chemokine patterns, which would not be able to form as a result of the free diffusion of chemokines. Such functional chemokine patterns, either soluble or immobilized in vivo.Chemokine GPCRs may also exhibit the full spectrum of features characteristic of ACRs. In some settings chemokine GPCRs are uncoupled from G-proteins and downstream signaling e.g. in response to physiological or pharmacological stimuli Individual expression patterns of ACRs, also diverse ACR activities, including various outcomes of their interactions with chemokines and how these impact on chemokine functions, are discussed in some detail below.DARC was initially described as the Duffy (Fy) blood group antigen Plasmodium vivax and knowlesi use DARC to invade erythrocytes Plasmodium falciparum does not require DARC for erythrocyte invasion and therefore the Duffy negative phenotype does not protect its bearers from falciparum malaria. A rare C286T substitution in FY*B  leads to a “weak” Fyb expression Two malarial parasites, HIV-1 also binds to DARC on erythrocytes allowing them to transmit the virus to mononuclear and other susceptible blood cells The observation that erythrocytes of Duffy-positive but not negative individuals bind IL-8 led to the establishment of Duffy antigen as a chemokine receptor in situ and in vitro.In addition to chemokines, DARC was suggested to bind in trans-geometry CD82, a tetraspanin expressed by tumor cells E. coli pneumonia the overproduction of CXCL5, a chemokine with high affinity for DARC, resulted in saturation of erythrocyte DARC and inhibition of CXCL1 and CXCL2 binding In concert with early ideas about the function of ACRs, it has been assumed initially that the role of DARC is limited to negative regulation of chemokine activities. This was concluded based on the chemokine “sink” function ascribed to the erythrocyte DARC Surprisingly little is known on how distinct characteristics of chemokine homeostasis in Duffy-negative individuals impact on the development of human diseases. Nevertheless, there are correlates of Duffy negative polymorphism with incidences of benign ethnic neutropenia in vitro studies unequivocally established DARC as a transcytosis receptor that transports chemokines unidirectionally, from the baso-lateral to apical side only, but not vice versain vitro and in vivoLeukocyte egress from blood occurs predominantly in venules and small veins where chemokines can be immobilized on the luminal membrane of endothelial cells D6 is an ACR for at least twelve inflammatory CC chemokines and has a high homology with their classical GPCRs A characteristic feature of almost all D6 ligands is a proline in N-penultimate position in situ hybridization showed that in several of these tissues D6 is expressed by the endothelial cells lining afferent lymphatic vessels in situ chemokine binding studies In vitro stimulation of monocytes and macrophages with major pro-inflammatory cytokines leads to a decrease in D6 expression, whereas treatment with TGFβ results in its increase The mapping of D6 mRNA expression in human and mouse tissues revealed its presence in many organs and tissues including lung, liver, spleen, kidney, heart, muscle, brain, placenta, gut and skin in vivo studies probing the function of this ACR in different diseases. Under normal conditions D6 deficiency does not convey any apparent disadvantages. However, when challenged with various stimuli D6 knockout mice develop increased inflammatory responses. For example, following topical treatment with phorbol ester or Freund's adjuvant injection D6−/− mice show augmented skin inflammation as compared to wild type controls Mycobacterium tuberculosis infection D6 expression prevented the lethal outcome seen in D6 knockouts by suppressing the chemokine and cytokine storm and reducing leukocyte infiltration into the lungs and other organs The use of D6 knockout mice enabled an array of In placenta, an organ with the highest D6 expression, it is on syncytiotrophoblasts, cells covering placental villi However, not in all experimental models D6 plays an unequivocally protective role. D6 is expressed in the brain, primarily by astrocytes Much less is known about the contribution of D6 to human diseases. D6 is expressed in several human tissues and organs and is upregulated in different inflammatory and autoimmune diseases. However, it is not clear if and how such increased levels of D6 expression may contribute to the pathogenesis. Like all ACRs, D6 is expressed in human malignancies including angiosarcomas αiCXCR7 was originally identified as a GPCR from a dog thyroid cDNA Additionally, CXCR7 was also described on subsets of B cells Chemokine (CC-motif) receptor-like 1 (CCRL1) Absence of CCRL1 in mice leads to strong increase in blood CCL21 titers and increase in the levels of CCR7 ligands CCL19 and CCL21 in lymph nodes but not in spleen Chemokine (CC-motif) receptor-like 2 (CCRL2) is also known as L-CCR (LPS-inducible CC chemokine receptor related gene) in vivo phenomena and provide an important molecular bias to the pathophysiological processes that take us from the womb, through years of balanced wellbeing and via countless avenues of possible diseases, ultimately to the grave. Also, the early general perception of ACRs as savage chemokine decoys has been replaced by more deferential awareness and deeper understanding of their multiple and versatile functions. The recent discovery of the ACRs for homeostatic chemokines, progress in defining various functional facets of both “old” and “new” ACRs and mapping their expression in health and disease brought us much closer to deciphering “chemokinese”, the chemokine-based universal language of cell communication. Nevertheless, despite giant leaps in understanding ACRs, we are still not entirely sure about the grammatical rules of their involvement. Future studies should unravel plentiful obscure aspects of ACR biology including, but not limited, to the following issues. 1). Many but not all chemokines have been assigned a cognate ACR. The outstanding chemokines either may not require an ACR for their in vivo activities or their interactions with ACRs have not yet been uncovered. No comprehensive ligand mapping of ACRs has been attempted. This allows for a possibility that remaining chemokines may bind known ACRs. Alternatively, some of the orphan heptaspanning membrane receptors may be chemokine ACRs. It is clear that potential interactions of chemokines with putative ACRs cannot be unveiled using traditional methods of GPCR deorphanization, which rely on functional cell responses as readouts. Prospective binding studies of isotope- or fluorochrome-labeled chemokines to orphan heptaspanning receptors may uncover new chemokine ACRs. 2). It is not known what possible signaling pathways are initiated through ACRs and how broad is their spectrum for any individual ACR. It is also not clear if and how some of ACR-derived signals affect the availability and function of the intracellular intermediates downstream of GPCRs. Future systematic analysis of ACR interactomes may bring new understanding of the biochemical cascades unleashed by ACRs including those potentially harnessing GPCRs-mediated signals. 3). Upon their internalization, different ACRs complete dissimilar intracellular itineraries. To date it is not apparent which molecular cues allow ACRs to couple with alternative vesicular pathways. Yet unidentified functional moieties within the intracellular portions of ACRs may select putative secondary effectors and determine subsequent differential targeting into distinct endosomal compartments. Also, it is not clear how much the nature of ACR itself versus the subcellular makeup of any particular cell type affect the outcome of chemokine interactions with ACRs. Comparative functional studies of wild type and altered ACRs expressed in various host cells may answer these questions. 4). In addition to paradigmatic scavenging and transport, there may be other potential outcomes of chemokine binding by ACRs. These may include chemokine immobilization within functional microdomains of the cell membrane or nuclear targeting of chemokines. Morphological studies using high-resolution subcellular imaging may suggest such new ACRs activities. 5). Interrogating the functions of ACRs in knockout mice leaves questions open about the relative involvement of ACRs on different cells and at different sites as well as potential compensatory roles of other ACRs. The generation of conditional knockout mice, still outstanding for most ACRs, will allow selective ACR gene deletion in different organs and tissues. 6). On the whole, still very little is known about the exact contribution of ACRs to human diseases. Immunohistochemical mapping of ACR expression in human tissues has been challenged by the general lack of suitable specific antibodies recognizing ACRs in tissues. Potential discovery of polymorphic variants of ACRs with altered function and their correlations with courses and outcomes of human diseases would facilitate our understanding of different roles ACRs play in pathogenesis. This, in turn, should allow the design of new therapeutic strategies targeting ACRs for treatment of human diseases with significant chemokine contribution to their pathogenesis. Such approaches may involve, depending on the context, either interference with pathological ACR expression and function or, on the opposite, the induction and up-regulation of ACRs expression.The studies performed during the last several of years and reviewed here helped to propel ACRs from the fringes of chemokine research into its mainstream. It is clear now that ACR activities contribute to the majority of chemokine-driven


Vascular smooth muscle cells have attracted considerable interest as a model for a flexible program of gene expression. This cell type arises throughout the embryo body plan via poorly understood signaling cascades that direct the expression of transcription factors and microRNAs which, in turn, orchestrate the activation of contractile genes collectively defining this cell lineage. The discovery of myocardin and its close association with serum response factor has represented a major break-through for the molecular understanding of vascular smooth muscle cell differentiation. Retinoids have been shown to improve the outcome of vessel wall remodeling following injury and have provided further insights into the molecular circuitry that defines the vascular smooth muscle cell phenotype. This review summarizes the progress to date in each of these areas of vascular smooth muscle cell biology. Cell lineage determination and differentiation are essential for the normal development of the human body. Cellular identity is hard-wired in an array of signaling pathways that converge upon the nuclear genome to orchestrate proper patterns of gene expression requisite for the specific functions performed by the ∼250 distinct cell types. Thus any given cell type's identity and phenotypic characteristics are determined primarily by the signaling input, both intrinsic and extrinsic, and the subset of nuclear genes that are transcriptionally and post-transcriptionally controlled through such signaling events. One cell type of paramount importance in the developing and postnatal body plan is the vascular smooth muscle cell (VSMC). These cells are derived from a variety of distinct regions in the developing embryoMYH11), alpha actin (ACTA2), gamma actin (ACTG2), and calponin (CNN1) as well as SM22 alpha (TAGLN) and smoothelins (SMTNA and SMTNB) all show adult VSMC-specific patterns of expression. Interestingly, however, many of these genes show transient expression in developing skeletal and/or cardiac muscleThe positioning of VSMC within the tunica media of blood vessels offers a histological definition of this cell type. However, like all human cell types, VSMC may also be defined in molecular terms by the expression of a subset of the > 23,000 genes in the human genome. Over the last 25 years, major progress has been made in defining the unique transcriptome of VSMCe.g., MYH11) can undergo alternative splicing to generate functionally distinct proteins. Such splicing events likely explain how such a complex organism as a human being is endowed with only a marginal increase in gene number over more simple animals . Some VSMC-specific genes arise from alternative splicing of more widely expressed genes. The latter include the alpha tropomyosin gene (TPM1), telokin (MLCK_v7), and heavy caldesmon (CALD). VSMC-specific splice variants likely require specialized components of the spliceosome found only in the VSMC lineage. In sharp contrast to the transcriptional events underlying VSMC-specific gene expression, the nature of VSMC-specific splicing is only marginally understoodThe majority of genes in the human genome undergo alternative splicingA myriad of signaling pathways has been described that positively regulate the VSMC differentiated phenotype. One strong stimulus for VSMC gene expression is stretch due to the increasing pressure exerted by the heart on the vascular treecis element known as the CArG boxFOS, which is known to be associated with the growth response of many cell types including VSMCMost signal transduction pathways converge upon the nucleus to direct cell-specific gene expression through the action of DNA-binding transcription factors and a litany of co-regulators that are recruited to discrete elements bound by the signal-responsive transcription factor. In VSMC, as in cardiac and skeletal muscle, the principal DNA-binding transcription factor involved in cellular differentiation is serum response factor (SRF)The paradigm for differentiation of a multi-potential cell to one of more definitive nature carrying out specific functions to maintain homeostasis was established with MyoD in skeletal muscle. These seminal findings, first described by Andrew Lassar in the late Harold Weintraub's labMYOCDe.g., FOS and EGR1) versus cardiac muscle genes; MYOCD does not effect growth-related gene expression. A subsequent study extended MYOCD's expression to cultured VSMC and adult aortic tissue and, predictably, this SRF cofactor greatly enhanced VSMC-specific promoter activity where CArG elements residecis element, though the atomic structure of MYOCD bound to SRF over a CArG element has yet to be solved. Since the first description of MYOCD as a molecular switch for the VSMC differentiated phenotypeDazhi Wang, in the laboratory of Eric Olson, sifted through EST databases for genes uniquely expressed in cardiac muscle and found, among many, a gene initially thought to be restricted only to cardiac muscle, hence its designation as in vitro and shown to exhibit accelerated growth with the loss of contractile propertiesin vivo when blood vessels were subject to mechanical or dietary injuryin vitro or in vivo following arterial insult, is not clear since the promoter to MYOCD is very complex. Recently, however, evidence has emerged for an important role of miR-145 in MYOCD regulation. Just as MYOCD is down-regulated under conditions favoring VSMC phenotypic modulation, miR-145 expression levels are greatly reduced after mechanical injury or diet-induced atherosclerosisFor more than 40 years, VSMC biologists have struggled to understand the basis for so-called VSMC phenotypic modulation. This process was initially described, inappropriately, as “de-differentiation” when VSMC were cultured MLCK_v7 (telokin) promoter activity and expression (which is low in VSMC) or synergize with MYOCD and activate MYH11 promoter activity. In addition, GATA6 turns on other VSMC contractile genesMYH11 promoter region and increase endogenous expression of some VSMC-specific contractile genesMYOCD expression levels were also compromised suggesting that c-MYB either directly or indirectly activates the MYOCD gene. Finally, a very recent study has shown that the NRF3 transcription factor, involved with redox control in a cell, positively enforces the VSMC contractile phenotype, in part, through the elevated binding of SRF and MYOCD to CArG elements as well as the up-regulation of MYOCD itselfAlthough MYOCD is without question, the critical factor for the establishment and maintenance of VSMC differentiation, there are additional transcription factors that play some role in this process as well. For example, GATA6 was shown to be down-regulated in the injured vessel wall concomitant with reduced contractile genes. Adenoviral-mediated gene transfer of GATA6 to the injured vessel wall restored contractile gene expression and reduced vascular neointimal formatione.g., Am80) to animals subjected to balloon angioplasty or an atherosclerotic regimen showed reductions in vascular occlusive diseaseEven before MYOCD was discovered, labs world-wide were exploring ways to prevent VSMC phenotypic modulation, and by extension vascular disease, in an array of vascular injury models. One such foray involved the study of retinoids, which are natural and synthetic derivatives of vitamin A that act as ligands for nuclear transcription factorsin vitro and in vivo studies of retinoids in the vessel wall prompted a screen for retinoid-response genes. Using a modified subtractive hybridization assay wherein cultured VSMC stimulated with or without ATRA, 14 novel retinoid-response genes were identified that showed either immediate early responses to ATRA that did not require de novo protein synthesis or delayed responses requiring new protein synthesis for inductionRetinoids exert their biological actions primarily through the activation of nuclear receptors that upon ligand activation, direct changes in gene expression. There are 6 retinoid receptors, three retinoic acid receptors (RAR) that mainly bind ATRA and three retinoid X receptors (RXR) that mainly bind the 9-cis stereoisomer of ATRATGM2) exhibited very robust and early activation with ATRA and other stereoisomers of ATRAin vivo may involve an element of apoptosisThere are at least four transglutaminase genes whose encoded proteins play critical roles in the cross-linking of proteinsITGA8)ITGA1, ITGA5, and ITGB1The superfamily of integrin genes are involved with diverse biological properties such as growth, differentiation, and outside-in signaling events. Application of ATRA to cultured VSMC resulted in the delayed induction of alpha 8 integrin  and downstream substrates of the effector proteinIn order for generic signaling to confer varied responses across each of the some 250 cell types, cells must compartmentalize signaling in a manner that best befits the cell's homeostatic balance. One manner in which cells do this is through the action of A-Kinase Anchoring Proteins (AKAPs) that can bind both effector protein  is largely known for its important role in endothelial cells during fatty streak formation in early atherogenesis, this marker of inflammation is also known to be expressed in VSMC. In fact, the initial knockout of VCAM-1 exhibited a defect in VSMC differentiationD9. Initial Northern blotting studies revealed that D9 is a small transcript (less than 1 kb) suggesting that it could represent a novel microRNA. Attempts to clone other orthologs of D9 from other species have been unsuccessful and screening of a rat genomic library failed to uncover a genomic clone. Remarkably, sequence analysis of D9 has failed to reveal any homology to anything in all of the annotated databases. At this period of time, we are completely at a loss as to whether D9 represents an unknown protein coding gene, a non-protein coding RNA gene or something vestigial to an infection of the rat genome from which we derived the initial VSMC for the retinoid-response gene screen.One of the “genes” found to be induced with ATRA in VSMC goes by the provisional name of RISC due to its amino acid homology to a large family of plant serine carboxypeptidases bearing a classic catalytic triad comprising the amino acids serine, aspartic acid, and histidineSCPEP1) because of the emerging field of RISC biology and more importantly, the subsequent finding that SCPEP1 is not induced by retinoids. Nevertheless, SCPEP1 is expressed in VSMC and across multiple tissues as shown by antibody studiesSCPEP1 does not show an overt phenotype as mice survive and breed without any histological evidence of pathologySCPEP1 null mice show reduced neointimal load suggesting that SCPEP1 directs VSMC migration and proliferation. Indeed, adenoviral delivery of SCPEP1 to VSMC causes accelerated growth and migration in a catalytic triad-dependent mannerOne of the retinoid-response genes originally cloned was a novel gene we called e.g., eluting stents) is unclear at the moment. One would think that given the years of clinical experience with retinoids for the treatment of cancer and dermatopathologies, that an application in the setting of certain vascular diseases such as recalcitrant transplant arteriopathies would be warranted.Several other retinoid-response genes in VSMC exist that await further study. These include endolyn, ceruloplasmin, importin alpha, cathepsin-L, and an unusual transcription factor called SALF that results from the fusion of two co-transcribed genesA series of scientific revolutions has occurred in only the last 57 years since the elucidation of the structure of DNA


Gastric cancer is the second most lethal cancer world-wide, and has a poor overall 5-year survival rate of <25% which is largely due to both late detection of this aggressive disease and the limited effectiveness of current treatment options. Surgery alone can only “cure” a small proportion of patients with locally-invasive advanced gastric cancer, and while adjuvant perioperative chemotherapy  reduces tumour sizes and improve the 5-year survival rate , patientGastric cancer represents an increasing number of cancers, including colon, liver, lung and pancreatic, whereby disease pathogenesis is intimately linked with chronic inflammation . It is therefore perhaps not surprising that over recent years there has been an explosion in research to identify key regulators of the immune system with oncogenic potential that promote the transition from a chronic inflammatory state to one of carcinogenesis. In this respect, much attention has focussed on pathogen recognition receptors, in particular members of the Toll-like receptor (TLR) family which act as critical sensors of the immune system to trigger the inflammatory response to many microbial  insults . SupportTLR2 gene in tumours of advanced gastric cancer patients is associated with poor overall survival [A recent study by Tye and colleagues has paved the way for new insights into this issue, based on the discovery that the specific and augmented expression of the survival . Furthersurvival .The identification of TLR2 as a new gastric cancer gene now paves the way for several key avenues of research. For example, pinpointing specific molecular pathways acting downstream of TLR2 critically responsible for gastric carcinogenesis is an important question, which could be potentially addressed through the identification of gene signatures triggered by TLR2 activation in gastric epithelial tumour cells, and mapping these TLR2 signatures to databases of other known pathway activation profiles. The availability of a robust disease-associated TLR2 signature could also facilitate patient stratification efforts in selecting those specific gastric cancer patients that are most likely to respond to a TLR2-targeting therapy. We also propose that future research efforts be directed towards examining the genomes of primary gastric tumours to identify somatic alterations in TLR2 (either mutation or gene amplification) that might cause hyper-activation of TLR2, analogous to the epidermal growth factor receptor in lung cancer.In addition to its newfound role in gastric cancer, TLR2 has been implicated in numerous other inflammatory diseases and cancers, such as lung and pancreatic cancer, arthritis, and ischemia reperfusion (I/R) injury, which collectively make TLR2 an attractive therapeutic target. In this regard, the murine monoclonal antibody OPN301 used by Tye and colleagues to effectively suppress gastric tumour growth is also efficacious in pre-clinical models of kidney transplantation, cardiac I/R injury and pancreatic cancer . Based oin vitro, including short interfering RNA (siRNA), small molecule inhibitors identified in cell based screening assays, and peptide mimetics that prevent ligand-induced receptor signalling of the intracellular domain of TLR2. While the anti-tumour efficacy of such strategies in vivo remains to be explored, a recent in vivo study successfully used TLR2-specific siRNA to block tumourigenesis in a xenograft model of liver cancer [We do note, however, the existence of additional approaches to inhibit TLR2 activity r cancer .Our recent study suggests that at least in the context of gastric cancer, TLR2 antagonists can ameliorate tumour growth by acting directly on the tumour cells to promote apoptosis and suppress proliferation . However


The number of children with mild neurodevelopmental disorders, such as autism, has been recently increasing in advanced countries. This increase is probably caused by environmental factors rather than genetic factors, because it is unlikely that genetic mutation rates suddenly increased within a short period. Epigenetics is a mechanism that regulates gene expression, depending not on the underlying DNA sequence but on the chemical modifications of DNA and histone proteins. Because mental stress can alter the epigenetic status in neuronal cells, environmental factors may alter brain function through epigenetic changes. However, one advantage of epigenetic changes is their reversibility. Therefore, diseases due to abnormal epigenetic regulation are theoretically treatable. In fact, several drugs for treating mental diseases are known to have restoring effects on aberrant epigenetic statuses, and a novel therapeutic strategy targeting gene has been developed. In this review, we discuss epigenetic mechanisms of congenital and acquired neurodevelopmental disorders, drugs with epigenetic effects, novel therapeutic strategies for epigenetic diseases, and future perspectives in epigenetic medicine. All fouEpigenetic gene control is an intrinsic mechanism for normal tissue development and abnormalities in the molecules associated with this mechanism are known to cause various congenital diseases. It is notable that some congenital diseases with defects in epigenetic gene regulation show neurological features and mental retardation.Genomic imprinting is the initial epigenetic phenomenon discovered in human diseases. In an imprinted gene, one of the two parental alleles is active and the other is inactive due to an epigenetic mechanism such as DNA methylation A. TherefThe X chromosome has a large number of genes relative to the Y chromosome. Thus, females (XX) have more genes than males (XY). To minimize this sex imbalance, one of the two X chromosomes in females is inactivated by an epigenetic mechanism . ImpropeEven when failure of X-chromosome inactivation occurs in women with one normal X chromosome and a small X chromosome due to a large terminal deletion and thus over dosage effect of X-linked genes is small, such affected women show severe congenital neurodevelopmental delays ,15, indi3) to CpG dinucleotides.A defect in one of the DNMTs  can cause an ICF syndrome that is characterized by immunodeficiency, centromere instability, facial abnormalities, and mild mental retardation , which is also known as the nuclear receptor subfamily 3 group C member 1 (NR3C1), increased in the hippocampus, which suppressed expression of the gene within the first week of life, whereas promoter methylation decreased in the brains of the offspring of high maternal care mothers during the same period [NR3C1 promoter in combination with a decreased level of its expression [Short-term mental stress after birth may alter the epigenetic status in the brain and result in persistent abnormal behavior  Figure .In rat pe period . This stpression . This fipression , and alsIn cancer, it has been established that certain tumor types respond well to DNMT and histone deacetylase (HDAC) inhibitor treatments, with the best clinical efficacy seen in hematologic malignancies . HistoneBdnf) in the hippocampus [It was recently shown that the major antidepressant drug imipramine reverses the depressive state by altering an epigenetic mark (histone modification) in the brain-derived neurotrophic factor gene mouse embryos exhibiting neural tube defects [Folic acid is an important nutritional factor and a mediator in the transfer of methyl groups for DNA methylation. Folic acid enters the pathway for synthesis of methionine via dihydrofolate and can be recycled through the formation of tetrahydrofolate (THF). THF is converted to 5,10-methylen-tetrahydrofolate  and 5,10 MTHF is reduced to 5MTHF. 5MTHF then remethylates and converts homocysteine to methonine; methionine is converted to S-adenosyl-methionine (SAM), which is the major methyl donor for DNA and cellular methyltransferase reactions. Therefore, dietary folic acid may influence the maintenance of DNA methylation. Nevertheless, in contrast to the increase in US because of government mandated folic acid supplementation of grains and grain-based food products , folic a defects .Pparα and GR (or Nr3c1) in the liver of juvenile [Pparα, GR (or Nr3c1), and the insulin receptor gene (IR), causing suppression of their expression, increased weight gain, a lower plasma b-hydroxybutyrate concentration and increased hepatic and plasma triglyceride concentrations [Besides epigenetic change observed in the neonatal period as described earlier, such change was also observed in the fetal period due to inappropriate supply of nutrients of the mother, which increases the susceptibility of the fetus to develop diabetes mellitus . In ratsjuvenile  and aduljuvenile , and foltrations . These fSince the 1980s, folic acid has been given in an empirical fashion to autistic children and to adults with mental diseases; several studies indicate that this treatment may be effective in a subset of patients ,56,57. ADnmt3 during larval development has a royal jelly-like effect on development [In honeybees, female larvae fed with the secretion known as royal jelly develop as fertile queens. By contrast, their genetically identical siblings that do not receive royal jelly develop as sterile workers. Comparison of the queen and workers show that they have widespread differences in their methylation patterns. Silencing of the DNA methyltransferase gene elopment . Moreoveelopment .Epigenetic regulation is based on the attachment of modifiers, such as methyl residues, to the DNA or histone proteins; there is considerable speculation that this epigenetic mechanism can be modified in a controlled fashion by altering of the patterns of attachment and by removing such modifiers. If this proves to be the case, then epigenetically determined abnormalities in humans are in principal reversible, and therefore, potentially treatable.Mecp2 knockout mice illustrate the reversibility and treatability of neurological symptoms caused by an epigenetic failure. Mecp2 knockout mice mimic the neurological symptoms seen in patients with Rett Syndrome, including seizures, ataxic gait, and hind-limb clasping [Mecp2 gene into a Mecp2-knockout mouse [Mecp2 was initially silenced by an inserted stop codon, and then reactivated by treatment with tamoxifen . This treatment causes the Cre-estrogen receptor fusion protein to translocate from the cytoplasm, where it is inactive, to the nucleus, where the Cre recombinase acts to recombine the two loxP sites that flank the inserted stop codon. The mice exhibited neurological symptoms shortly after birth; however, after treatment with tamoxifen, the symptoms were much milder and the mice survived longer than first-generation Mecp2 knockout mice. These results indicate that the developmental absence of MeCP2 does not irreversibly damage neurons and that the subsequent neurological defects can be reversed. The results also indicate that neurodevelopmental disorders caused by abnormalities in epigenetic regulation, are potentially treatable after birth, and that a similar reversibility of neuronal damage might be expected not only in this DNA methylation-based epigenetic disease (Rett syndrome), but also in histone methylation based diseases [MECP2 gene into the patients prenatally, chemicals that activate the expression of MECP2 might work in patients with Rett Syndrome, since Rett syndrome patients  have one normal MECP2 besides one mutant MECP2.Recent studies on clasping . A new Mut mouse . To proddiseases ,62,63. AUbe3a) is silenced and only the maternal allele is active. In a mouse model in which the maternal allele is defective , topoisomerase inhibitors have been shown to activate the silenced paternal Ube3a allele in neurons by reducing the transcription of an imprinted antisense RNA [Another potential epigenetic therapeutic strategy has been developed for the imprinting disorder Angelman syndrome. In normal mice, the paternal allele of the ubiquitin-protein ligase E3A gene  polyamides. These are small synthetic molecules that recognize and attach to the minor groove of DNA, thereby inhibiting gene transcription by blocking transcription factors that bind to the DNA in a sequence-specific manner . It has Mecp2 knock-out mice by altering gene expression and synaptogenesis in the brain [Mecp2308/+ female mice model) was more susceptible to a specific chemical  and showed impaired learning and long-term memory compared with a control mouse, indicating that aberrant epigenetic status leads to susceptibility to environmental factors [In addition to chemical treatments, recent mouse experiments also showed that appropriate environmental conditions  was largely disregarded following the acceptance of Charles Darwin’s theory of natural selection as the driving force of evolution. However, the recent advances in our understanding of epigenetic inheritance may, to a limited extent, lead to the resurrection of Lamarckian inheritance. Epigenetic marks allow the transmission of gene activity states from a cell to daughter cells. Initially, it was presumed that epigenetic marks were completely erased and re-established in each generation. However, several recent studies in some model organisms indicate that the erasure process may be incomplete at some loci in the genome, and that some epigenetic marks acquired in one generation can be inherited by the next generation. This phenomenon is termed “transgenerational epigenetic inheritance” ,73,74,75The transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germ line. In the rat, exposure of pregnant females to vinclozolin, an endocrine disruptor, caused infertility in some male offspring and a reduced rate of spermatogenesis in others; moreover, these effects were passed to the male offspring in subsequent generations . The DNACrfr2) and altered behavior patterns [One such study showed that mental stress in offspring due to maternal separation not only changed the DNA methylation status in the brain but also in the sperm, and the changed methylation status seen in the brain was accompanied by an altered expression of the corticotropin releasing factor receptor 2 gene  in the lymphocyte genomes of heavy tobacco smokers, which might affect their future cardiovascular function [The methodology for epigenomic analysis has developed from the single-gene level  to the cfunction . Recent function . Based ofunction .Understanding the human epigenome is fundamental to the investigation of congenital and acquired diseases, and will provide epigenetic biomarkers to which new drugs are targeted.


In plants, sucrose synthase (Sus) is widely considered as a key enzyme involved in sucrose metabolism. Several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, while limited information of Sus genes is available to date for cotton.GaSus1 to 7) identified from diploid fiber cotton (Gossypium arboreum). Comparisons between cDNA and genomic sequences revealed that the cotton GaSus genes were interrupted by multiple introns. Comparative screening of introns in homologous genes demonstrated that the number and position of Sus introns are highly conserved among Sus genes in cotton and other more distantly related plant species. Phylogenetic analysis showed that GaSus1, GaSus2, GaSus3, GaSus4 and GaSus5 could be clustered together into a dicot Sus group, while GaSus6 and GaSus7 were separated evenly into other two groups, with members from both dicot and monocot species. Expression profiles analyses of the seven Sus genes indicated that except GaSus2, of which the transcripts was undetectable in all tissues examined, and GaSus7, which was only expressed in stem and petal, the other five paralogues were differentially expressed in a wide ranges of tissues, and showed development-dependent expression profiles in cotton fiber cells.Here, we report the molecular cloning, structural organization, phylogenetic evolution and expression profiles of seven Sus genes (This is a comprehensive study of the Sus gene family in cotton plant. The results presented in this work provide new insights into the evolutionary conservation and sub-functional divergence of the cotton Sus gene family in response to cotton fiber growth and development. Sucrose is the major form of photosynthate for export from the source leaves into cellular metabolism of most plants . The traArabidopsis and rice, both contain an Sus gene family with six distinct active Sus genes, suggesting gene expansion in higher plants during evolution [Lotus japonicus, at least six Sus genes are known to exist in the Sus gene family [. Studies on these Sus sequences and phylogenetic relatedness revealed that structural conservation and functional divergence occurred within the gene family during evolution. Members of the Sus gene family in many plant species are divergent in function and differentially expressed during plant development. For example, maize harbors a Sus family containing at least three distinct genes, Sh1Sus1 and Sus3. Sh1 is most abundantly expressed in developing endosperm, and has the dominant role in cell wall synthesis. Sus1 is expressed in a wide range of tissues, but plays a major role in starch synthesis [Sus1Sus2 and Sus3. Sus1 is ubiquitously and highly expressed in the developing seed. Sus2 is mainly expressed in older testas and leaves, while Sus3 is weakly expressed only in flowers and young testas. Furthermore, the lack of Sus1 activity in mutant seeds and root nodules could not be compensated by Sus2 and Sus3[Arabidopsis contains three major Sus gene classes with distinct but partially overlapping expression profiles, and the specific roles have been assigned for each gene by extensive studies of corresponding knockout mutants [Lotus japonicus and citrus, Sus genes have also been demonstrated to be expressed in tissue-specific and development-dependent patterns [Arabidopsis, have been well studied, our knowledge of cotton Sus genes, especially their evolutionary mechanisms and potential functions in fiber growth and development, needs to be well explored.The identification and subsequent characterization of the genes encoding plant sucrose synthase is the first step towards understanding their physiological roles and metabolic mechanism involved in different growth processes. Recent studies have revealed that Sus isozymes are encoded by a small, multigene family that comprises at least three Sus genes in the most plant species. With the completion of the genome sequence analysis, recent studies showed that model plant species, olution ,24. Simi family . Studiesnthesis ,27. The  and Sus3. Arabidomutants . In otheatterns ,28,29. ISS3 , which was isolated previously from an upland cotton fiber cDNA library and has been demonstrated to play an important role in ovule development and fiber cell initiation [Cotton fiber is not only the world's most important textile material, but also the ideal experimental system for studying the mechanism of cell development based on its single-celled profile . Cotton tiation . For theIn the current work, we reported the identification and characterization of seven Sus genes in cotton species and investigated their expression patterns at the transcriptional level. The analysis in this study mainly focused on the gene identification, evolutionary relationship, exon/intron organization and tissue-specific expression patterns of each member of the cotton Sus gene family. Therefore, our results obtained from this study will provide a foundation and framework for the further studies to gain a comprehensive understanding of the physiological roles of each cotton Sus gene in regulating the cotton plant growth, especially for the growth and development of cotton fiber.G. arboreum), rather than the allotetraploid cotton species, was used as a template, to avoid the laborious analysis of allelic sequence variation within the AT and DT sub-genomes. Eventually, five distinct non-allelic genomic fragments exhibiting substantial homology to plant Sus genes were identified by massive sequencing of the PCR-generated clones. In addition, using the mRNA sequences of Arabidopsis Sus genes as queries, the cotton EST database was extensively searched, leading to the assembly of other two additional contigs as putative Sus genes. The seven newly isolated gene fragments were named as GaSus1 to 7, respectively, according to the naming convention for Sus genes in other plant species. 5' and 3' RACE subsequently resulted in the cloning of the full-length cDNAs of these genes, except GaSus2, of which the mRNA could not be detectable in all tissues of cotton (described later).In order to identify the potential Sus homologues in cotton, comparisons between coding sequences of plant known Sus genes were performed and regions exhibiting somewhat similarities were picked out and used to design multiple pairs of degenerate primers for PCR amplification. The genomic DNA of diploid fiber cotton (http://www.ebi.ac.uk/interpro/), two conserved, sucrose synthase and the glucosyl-transferase domains, which have been suggested to be typical signatures of Sus proteins were also identified in all the cotton GaSus genes  with their isoelectric point calculated to lie between 6.26 and 7.27 , compared to the other paralogues  of GaSus genes, seven genomic DNA sequences, encompassing putative overall coding regions of GaSus1 to 7, were cloned from the G. arboreum genome. A comparison between the cDNA (except GaSus2) and genomic sequences, revealed that these GaSus genes are interrupted by multiple introns. As shown in Figure GaSus1, GaSus3 and GaSus7 all contain 11 introns in their coding regions (between the start and stop codons), while GaSus4 and GaSus6 are characterized by one and three more introns  than the three paralogues. However, GaSus5 is characterized by having only 9 introns. As the absence of the cDNA sequence, the exon/intron structure of GaSus2 was predicted online using the FGENESH algorithm (http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind), which resulted in the definition of 10 putative introns within its putative coding regions. The last intron, with a length more than one kb, was predicted to be the largest intron located in the cotton Sus gene family. Comparative analysis of these introns indicated a marked greater difference in sequence similarities compared to that shown by the exons in GaSus genes (data not shown). In addition, most of the intron sizes of the seven GaSus genes range within 70–110 bp in length, except GaSus6, within which 57% (8 of 14) introns are larger than 120 bp in length  to the ATG start codon. In addition, all the introns confer the GT-AG splicing rule, except for the sixth intron of GaSus7, which was spliced at the unusual GC-AG splicing sites.Comparative analysis of the exon/intron gene structure may provide some clues for the understanding of the evolutionary mechanisms underlying the genesis of family genes. In order to investigate the exon/intron structure of the cotton Arabidopsis and rice. Moreover, GaSus7 exhibited an extension of the last exon compared to the other GaSus genes, such feature are also observed, but more conspicuous in the Arabidopsis AtSus6 and the rice OsSus6 orthologues. Alignments of both the cDNA sequences and their predicted amino acid sequences between GaSus7, AtSus6 and OsSus6 revealed poor identities in the extended regions of the last exons, but much high identities in the other coding sequence (data not shown). Moreover, similarity searches with the BLAST algorithm against the NCBI database using the extended regions of the three orthologues failed to hit any sequence, except themselves, that showed significant similarities. In addition, careful scrutiny of the exon/intron structure in coding regions of Sus genes from two dicots (cotton and Arabidopsis) and two monocots (rice and maize) revealed that among these 21 sequences, introns were present at a total of 16 positions, in which 14 introns, in conserved positions, were observed to be contained in most of the Sus sequences from the four distantly related species, suggesting that ancestral Sus genes common to monocots and dicots contained 14 introns in these positions. The presence of an intron in the 5' untranslated region of cotton GaSus3 sequence and introns 15 and 16 at novel positions in the Arabidopsis AtSus5 and rice OsSus5 sequences may represent derived characters during evolution. Several of the Sus genes lack one or more introns , G. sturtianum (C1) and G. raimondii (D5), as well as from an outgroup species Gossyploides kirkii (K). Sequence comparison and gene structure prediction of the orthologous genes from the other four diploid cotton relatives revealed identical amino acid sizes  to determine the robustness of the phylogram's topology. The phylogenetic tree analysis revealed both relatively deep evolutionary root and the existence of more recent duplications for the Sus genes. As shown in Figure GaSus genes (GaSus1 to 5) fallen into this dicot group, and cluster together by forming an independent cotton clade to the exclusion of citrus, Arabidopsis, as well as other dicots genes, suggesting that a single Sus gene has expanded through independent duplication within the cotton lineages occurred after cotton separation with citrus and Arabidopsis species. Within the cotton clade, three paralogues, GaSus1, GaSus3 and GaSus4, which share a very high degree of sequence similarities, grouped more closely in the phylogenetic tree. The short branches separating the three genes suggesting that duplications that gave rise to them occurred relatively recently. In addition, GaSus5 was found to be unambiguously distant from the other four paralogues within this group. In contrast to dicot Sus I group, the monocot Sus I group thus includes members exclusively from monocot species, in which the proteins can be divided into two subclasses. Both of the subclasses contain Sus genes from rice, maize, wheat, barley and bamboo, suggesting that some ancestor Sus genes had diverged before gramineous species evolved into different plant lineages, and then underwent independent evolution within each lineage.The dicot Sus I group contains the proteins solely from the dicotyledonous plant species, in which they are subgrouped by phylogeny. For example, one group of solanaceous Sus proteins, including members from tomato and potato species, forms a cluster together with the two Daucus carrot proteins, DcSus1 and DcSus2. These proteins are part of the largest group that includes brassicaceous, daucus, solanaceous, leguminous, populus and citrus proteins but not a single cotton proteins. For cotton, five of the seven GaSus6 and GaSus7, were evenly separated into the other two groups, Sus II and III, with members from both dicot and monocot species. Additionally, in these two groups, genes from dicots and monocots were not found in distinct subgroups, suggesting that the significant duplications that gave rise to Sus II and III groups should be no younger than the monocots/dicots split.Unlike cotton Sus genes expanded in dicot Sus I group, the other two paralogues, GaSus genes were firstly examined in various cotton tissues, including roots, stems, petals and young leaves, as well as fibers at different developmental stages using semi-quantitative RT-PCR. It is worth to point out that there is a very high sequence similarity within the encoding sequence of some GaSus genes, and their reverse primers were therefore designed within their 3' untranslated regions to guarantee the primer specificity. As shown in Figure GaSus2, of which the transcripts was undetectable in all tissues examined, and GaSus7, of which the transcripts could only be detected in stem and petal, the other GaSus transcripts were detected in a wide range of tissues and showed distinct but partially overlapping expression patterns, suggesting that Sus genes might be implicated in a range of physiological processes in cotton plant.Systematic analysis of the expression patterns of Sus family genes can help to reveal their possible physiological functions involved in different growth processes in plant. In order to better learn the potential functions of specific isoforms of Sus in cotton, the tissue-specific expression of GaSus gene in different tissues and in fibers at different developmental stages , SusBSusC and SusD, had been identified in the tetraploid upland cotton species [Recently, benefiting from the whole genome sequencing of model plants, dozens of genes encoding Sus isozymes have been identified from various plant species using the comparative genome approaches. Typically, the complete genomic sequence of the model species species . Our preGaSus genes and other plant homologues in our work corroborated this classification , and further support the idea that higher plant species may have at least one gene for each of the three groups [GaSus1 to 5, in the Sus I group that cluster together with other dicot genes and separately from multiple monocot Sus genes is the direct evidence of that multiple gene duplication events occurred after the monocot/dicot divergence both within dicots and monocots, giving rise to two subclades: dicot-specific Sus I group and the monocot-specific Sus I group. As the five cotton genes group together to the exclusion of citrus, Arabidopsis and other dicot homologues in our phylogenetic tree, it is suggestive of that gene duplication events that gave rise to the five cotton Sus genes occurred after the separation of cotton/citrus/Arabidopsis from a common ancestor. The presence of one cotton Sus gene in each of the other two groups, Sus II and III, with members from both dicot and monocot species, indicating that Sus II and III groups containing GaSus6 and GaSus7 are evolutionarily older than dicot Sus I group containing GaSus1 to 5.Although the evolution of multigene families involves multiple mechanisms, comprehensive analysis of phylogenetic tree and exon/intron gene structures, to a certain extent, allow us to make some generalizations and predictions about the possible origin of and relationships between different isoforms of Sus, as well as their possible function. Plant Sus proteins have been historically divided into at least three major groups  on the basis of phylogenetic tree and molecular structures analysis of their sequences ,29. Phyl groups . The preArabidopsis, without exception, have lost introns 5 and 12. Since that members from other dicots, such as Leguminosae Lotus japonicus and Rutaceae citrus, of this group also have lost the two introns [GaSus1 to 5), one relatively earlier event of concerted intron loss of introns 5 and 12 occurred immediately after the dicot/monocot split but predated the separation of cotton and most of the other dicots. After this event, independent events of intron loss of introns 2, 4, 6, 11 and 13 occurred related to different dicots members in Sus I group. A comparison of the number and density of introns in the gene structures of the five cotton paralogues belong to the Sus I group further demonstrated that GaSus5 was produced from relatively more ancient duplications in comparison with its three paralogues GaSus1GaSus3 and GaSus4. Intriguingly, although Sus II group is thought to be evolutionarily older than dicot Sus I group, members in Sus II group appear to have underwent a relatively slower evolutionary rate, since that exon/intron structures of dicot genes (including cotton GaSus6) in this group have greater similarity to those of monocot Sus genes and also to the putative ancestral Sus genes  or additional exons (AtSus5LjSus6 and OsSus5) in their 3' regions, it is tempting to speculate that a sequence with at least two introns had been introduced to the 3' region of the progenitor of Sus III group probably by an ectopic recombination that occurred before the monocots/dicots splits. The absence of commonalities on sequence and exon/intron structure in the extended regions among different members may be due to that the extended region did not involve in the definition of the protein activity of Sus , confirming that AtSus5, but not AtSus6, is the orthologuous gene of cotton GaSus7. This is also indicative of that the loss of intron 1, 6 and 12 from cotton GaSus7 were the events that occurred before the separation of cotton and Arabidopsis lineages.It has been well demonstrated in many duplicated gene families that exon structure in homologues genes may even be conserved despite low sequence conservation . Differeintrons ,29, it tSus6 and Sus7. A second duplication that occurred also predated the monocots/dicots split was associated with the arising of the precursor of Sus1 to 5. Following the divergence from a common ancestor of citrus and Arabidopsis, a duplication occurred in cotton lineages and giving rise to Sus5, which subsequently underwent independent evolution and retained as a single gene, while its duplicate has expanded into four paralogues (Sus1 to 4), among which, GaSus3 and GaSus4 were produced from the most recent duplication events and consequently became the closest related genes in cotton Sus family, their closeness is well reflected in the terminal subgroups of the phylogenetic tree  has played a crucial role in driving evolutionary novelty that allow organisms to differentiate new organs or increase fitness to new environments ,51. To dSus genes occurs in cotton, as did the members of this Sus family in several other plant species, such as Arabidopsis[Lotus japonicus[In this study, we demonstrated that differential expression of the abidopsis,23, riceabidopsis, Lotus jjaponicus, citrus japonicus, and Bamjaponicus. The difjaponicus.GaSus genes in cotton ovules and fibers , an orthologous gene of GaSus3, in tetraploid upland cotton, repressed cotton early seed development, fiber cell initiation and elongation. In contrast to GaSus3 expression, the transcripts of GaSus5 were very low at 0 and 5 dpa, but increased continuously in abundance and reach its highest level in fibers around at 15 dpa, and then a significant decrease as the fiber cells developed further, suggesting that GaSus5 may primarily participated in fiber cell elongation and primary cell wall synthesis. The differential, complementary expression profiles of GaSus3 and GaSus5 is the direct evidence of that specialized GaSus genes might have been evolved to meet the requirement of the carbohydrates for cellulose synthesis, fiber cell initiation and elongation during cotton fiber development. Interestingly to note, of the seven cotton Sus genes, GaSus7 is not only the oldest duplicated gene, but also is the only one which is transcriptionally active, but not expressed in ovule and fiber cells. The strong transcription level of GaSus7 in petal implies that GaSus7 may be primarily involved in the regulation of floral organs, but not the ovules and fibers development in cotton. As GaSus6, another old duplicated gene, is also lowly activated in cotton fiber cells compared to those relatively recent duplicated paralogues, such as GaSus1 and GaSus3 , which was obtained from USDA collection through Dr. Peng Chee , was used in this study for cloning Sus genes and gene expression analysis. The seeds were delinted, germinated and maintained in pot culture under natural conditions for three weeks, and then the plantlets were either harvested for collecting the roots, stems and leaves samples or transplanted to open field for continuing growing. For analysis of Sus gene expression during cotton fiber development, each flower was tagged on the day of flowering and were taken as 0 dpa (days post anthesis). Developing ovules were subsequently harvested at various developmental stages from 0 to 20 days after flowering, and fibers were carefully scraped from the epidermis of the ovules. The collected samples were immediately frozen in liquid nitrogen and stored at −80°C for subsequent RNA isolation.G. arboreum, DNAs used for gene structure deduction and phylogenetic analysis were from G. anomalum (B1) and G. sturtianum (C1) obtained from Dr Xinlian Shen , and G. raimondii (D5) and Gossyploides kirkii (K) provided kindly by Dr. Andrew Paterson (UGA), here Gossypioides kirkii was used as an our group species.Except G. arboreum). The PCR products obtained from both cDNA and genomic DNA amplification were cloned into the pMD19-T cloning vector , and then transformed into E.coli cells (DH5α) for massive sequencing. The obtained sequences were submitted to NCBI database for blast analysis. In addition, the cotton EST database were also searched using Arabidopsis Sus genes as queries to identify putative unidentified members belong to the cotton Sus gene family.Based on the sequences of plant sucrose synthase genes accessioned in GenBank, we designed multiple pairs of degenerate primers from the conserved regions for using to amplify the homologs in diploid fiber cotton . Approximately one and 10 μg high-quality total RNA were used respectively for the 3′ and 5′ RLM-RACE protocols, strictly following the manufacturer’s instructions.Arabidopsis. Multiple alignment of the nucleotide and deduced amino acid sequences were performed using the programs ClustalX (version 1.8) [http://www.lynnon.com/) with default gap penalties. A phylogenetic tree was constructed from amino acid sequences of deduced Sus proteins by neighbor-joining algorithms wrapped in the MEGA 5.0 phylogeny program (http://www.megasoftware.net).The sequence data used in this study were collected using the keyword “sucrose synthase” and a query search in the GenBank using the known Sus gene sequences from cotton and on 1.8) , and DNAhttp://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind). Predicted conserved domains were screened within the deduced amino acid sequences of corresponding Sus genes using the interproscan algorithm web server (http://www.ebi.ac.uk/Tools/pfa/iprscan/).Exon/intron structures analysis of the target Sus genes were either conducted by comparing the cDNA sequences and their genomic DNA sequences or predicted online using the FGENESH algorithm -PCR analysis, approximately two micrograms of DNA-free total RNA from each sample was used to synthesize first-strand cDNA in a 20-μl reaction solution using a M-MLV reverse transcription kit (TaKaRa), and the synthesized cDNAs were used as templates in the following RT-PCR reactions.UBQ7, was used as an internal standard to adjust the relative quantity of the cDNA of each sample used in the RT-PCR analysis.For each target Sus gene, PCR amplification was performed using Takara Taq polymerase with specific primer pairs listed in Additional File To confirm the specificity of each primer and also to exclude any genomic contamination in PCR amplification, each of the target genes was also amplified with the same PCR procedures from the genomic DNA, which contains at least one intron in its amplified products. The amplified fragments were examined by electrophoresis on a 1.5-2.0% (w/v) agarose gel and visualized by ethidium bromide (EB) staining.GaSus gene was standardized to the expression level of the cotton constitutive UBQ7 gene, calculated by the formula Y = 10-(ΔCt/3) . The specificity of primers designed for real-time RT-PCR  according to the Manufacturer's instructions. The amplification of the target Sus genes was monitored by SYBR-Green fluorescence signal every cycle. The Ct (cycle threshold) value, which was defined as the PCR cycle at which a significant increase of reporter florescence signal is detected instantly, was used as a measure for the starting copy numbers of the target Sus gene. Relative quantitation for expression level of each R Table  was confet al., .Sus: Sucrose synthase; EST: Expressed sequence tag; dpa: Days post anthesis.The authors declare that they have no competing interests.AQC and JKR contributed to the experimental design and manuscript drafting. QPL contributed to the manuscript editing. AQC, SEH and ZL performed the primer design, Sus gene cloning and RT-PCR validation. FFL, MQD and QPL performed the RNA extraction and bioinformatics analysis. All authors have read and approved the final manuscript.Amino acid sequences analysis of the cotton Sus genes.Click here for fileMultiple alignment of the amino acid sequences between three Sus genes GaSus1, GaSus3 and GaSus4.Click here for fileMultiple alignment of the DNA sequence between five orthologs of Sus7 cloned from four diploid cotton species G. arboreum , G. anomalum , G. sturtianum  and G. raimondii  and one outgroup species, Gossypioides kirkii . Corresponding regions shaded in colours represent sequence identities between these orthologous genes. The exon sequence is shown by single underline, and the intron sequences are shown by double underlines. The arrows upon the first intron sequence indicate the unusual GC/AG splicing sites in the Sus7 genes. The SSRs  in the five orthologs are boxed and shaded.Click here for fileGene-specific primers used for semi-quantitative RT-PCR amplification. The numbers out and in parentheses represent expected size of PCR products amplified from cDNA and genomic DNA, respectively.Click here for file


Age-related bone loss and osteoporosis are associated with bone remodeling changes that are featured with decreased trabecular and periosteal bone formation relative to bone resorption. Current anticatabolic therapies focusing on the inhibition of bone resorption may not be sufficient in the prevention or reversal of age-related bone deterioration and there is a big need in promoting osteoblastogenesis and bone formation. Enhanced understanding of the network formed by key signaling pathways and molecules regulating bone forming cells in health and diseases has therefore become highly significant. The successful development of agonist/antagonist of the PTH and Wnt signaling pathways are profits of the understanding of these key pathways. As the core component of an approved antiosteoporosis agent, strontium takes its effect on osteoblasts at multilevel through multiple pathways, representing a good example in revealing and exploring anabolic mechanisms. The recognition of strontium effects on bone has led to its expected application in a variety of biomaterial scaffolds used in tissue engineering strategies aiming at bone repairing and regeneration. While summarizing the recent progress in these respects, this review also proposes the new approaches such as systems biology in order to reveal new insights in the pathology of osteoporosis as well as possible discovery of new therapies. Bone remodeling is a physiological process that maintains the integrity of the skeleton by removing old bone and replacing it with young matrix. An imbalance between bone resorption and bone formation with ageing will result in the increased rate of bone turnover rate and bone loss. The age-related progressive bone loss is exaggerated in patients with osteoporosis, a disease characterized by decreased bone mass, increased bone fragility, and increased risk of fractures . As the κB ligand (RANKL), take effect mainly by reducing bone resorption. However, these therapies frequently exhibit secondary effects due to the coupling phenomenon of bone formation by osteoblasts and bone resorption by osteoclasts [In the case of the age-related bone loss or osteoporosis, the osteoblast-mediated bone formation is severely impaired , 2 due teoclasts . Therefoeoclasts , though In this paper, accordingly, we focus on the functional regulation of osteoblast lineage cells in health and osteoporosis, the currently proposed anabolic agents such as teriparatide (PTH(1–34)), sclerostin or DKK1 inhibitors, and strontium that target specific signaling mechanisms underlying the osteoblast function and osteoporosis. The wide use of strontium in various orthopaedic scaffolds is also summarized. Through the analysis of current knowledge, some insights into further studies of osteoblast regulation and therapeutic exploration are provided.Bone formation is dependent on the recruitment of sufficient number of osteoblasts and activity of individual osteoblasts. Osteoblastic cells are recruited to bone forming surfaces mainly from a group of skeletal stem cells with osteogenic differentiation potential. Bone marrow contains a small population of mesenchymal stem cells (MSCs) that are capable of giving rise to bone, cartilage, fat, or fibrous connective tissue . Cell po in vivo only ablates bone formation and osteoclastic bone resorption persists [Particularly, flattened bone-lining cells are thought to be quiescent osteoblasts that form the endosteum on trabecular and endosteal surfaces and underlie the periosteum on the mineralized surface. More primitive osteoblasts that are found near functioning osteoblasts in the bone remodeling unit can be identified based on their expression of alkaline phosphatase. Active and mature osteoblasts synthesizing bone matrix have large nuclei, enlarged Golgi structures, and extensive endoplasmic reticulum. These osteoblasts secrete type I collagen and other matrix proteins vectorially toward the bone formation surface. Besides, osteoblasts have several other important roles in bone remodeling, including production of osteoclastogenic factors and bone mineralization . Preostepersists . TherefoDuring bone formation, a subset of osteoblasts undergoes terminal differentiation and becomes engulfed by unmineralized osteoid . FollowiOsteocytes express osteocalcin, galectin 3, CD44, and several other bone matrix proteins that support intercellular adhesion and regulate exchange of mineral in the bone fluid within lacunae and the canalicular network. Osteocytes regulate phosphate metabolism and matrix mineralization through the secretion of phosphate-regulating factors such as FGF23, Phex, Dmp1, and expression of sclerostin (encoded by gene SOST) and DKK1 that negatively regulates Wnt and BMPs signaling . Osteocyβ (TGFβ), hedgehog families, Wnt signaling, and notch pathways [Differentiation of mesenchymal stem cells into the osteoblast lineage is under tight regulation orchestrated through multiple signaling pathways. Among the well-characterized are the fibroblast growth factor (FGF), transforming growth factor pathways .The FGF pathway consists of 23 ligands that transduce their signal through one of the four FGF receptors (FGFRs), functioning in initiating condensation of the mesenchyme and proliferation of progenitor cells. Upon binding to cell surface receptors, FGFs lead to activation of multiple signaling modules, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), STAT1, and PKC. Temporal expression and activity of FGFR are critical for membranous bone formation and regulate the proliferation, differentiation, and apoptosis of osteoblasts . FGFRs cβ superfamily. This group of proteins has a number of diverse functions in multiple developmental processes ranged from embryogenesis, organogenesis, bone formation, cell proliferation, and stem cell differentiation [β itself plays more complex role during bone remodeling, with the inhibition of Runx2 and osteoblast differentiation in vitro but mainly promoting bone formation in vivo [Bone morphogenetic proteins (BMP) are members of the TGFntiation –28. BMPsntiation –31. Indu in vivo , 32.β-catenin, an intracellular transducer. β-catenin is expressed in mesenchymal precursor cells and its inactivation promotes their differentiation into chondrocytes instead of osteoblast [β-catenin-dependent Wnt signaling, β-catenin is stabilized following binding to its receptors Frizzled (FZD) and lipoprotein receptor-related protein 5 (LRP5) or LRP6, which in turn lead to the transcription of target genes such as Runx2. On the basis of extensive studies conducted thus far on Wnt signaling in bone, the pathway has now become the target for therapeutic intervention to restore bone strength in millions of patients at risk for fracture [During skeletal development, the Wnt signaling is implicated in multiple steps and processes, including proximal-distal outgrowth and limb patterning, and in MSC lineage commitment for chondrogenesis, osteogenesis, myogenesis, and adipogenesis –36. Consteoblast . During fracture .The hedgehog signaling through Indian hedgehog (Ihh), a secreted molecule of the hedgehog family, is required for osteoblast differentiation through endochondral bone formation. Ihh binγ-secretase complex. Consequently, the intracellular domain of notch is released from the plasma membrane and translocates to the nucleus, interacting with RBPJκ/CBF1 to activate downstream target transcription factors. By physically associating with Runx2 and interfering with functional activity of Runx2, notch inhibits osteoblast differentiation. Mutations in the notch signaling cause skeletal patterning defects in human and notch deficiency leads to severe osteoporosis in mice [The notch signaling mediates broad cell-cell communications. Once their ligands are binding to the neighbouring cell surface, notch receptors are cleaved by the  in mice . Interes in mice . Thus faBesides local growth factors, a number of systemic hormones regulate bone mass by regulating osteoblast differentiation and influencing bone formation. Many of them act on osteoblasts to express M-CSF and RANKL that in turn regulate osteoclast differentiation. We herein only discuss some as examples, particularly those understood well thus far.Parathyroid hormone (PTH) produced by the chief cells of parathyroid gland plays a primary role in calcium homeostasis through its action on bone and kidney and through enhanced synthesis of another hypercalcemic hormone, 1,25(OH)2 vitamin D3 . The anaβ2-adrenergic receptors (β2-AR) on osteoblasts [β2-AR have increased bone mass but do not respond to leptin by reduction in bone mass [β2-AR null mice fail to lose bone mass, suggesting that maintenance of the sympathetic nervous system in bone may require estrogen, which is essential in regulating bone remodeling via two related receptors, ERα and ERβ expressed by osteoblasts.An important nonsteroidal regulator of bone mass is leptin, a hormone that functions through an inhibitory action of the hypothalamus on bone formation , 51. Lepeoblasts . Mutant one mass , while oα, and RANKL) and stimulate the secretion of OPG by osteoblasts [The correct balance between bone deposition and resorption is crucial for the proper maintenance of the bone mass and the loss of this coupling is the starting point for osteoporosis . This iseoblasts , 58.κB signal transduction in osteoclast development and functional activity is widely recognized [κB activation is potently inhibitory to osteoblast commitment, differentiation, and mineralization in vivo and in vitro [α) is a potent NF-κB inducer and activation of p65 (a NF-κB subunit) by TNF-α has been shown to suppress transcription of osteocalcin in osteoblastic cells [α is reported to reverse age-related defects in bone formation in a mouse fracture healing model and synergizes TGFβ- and BMP-2-induced Smad signaling in differentiating osteoblasts [α upregulates Smurf1, an E3 ligase that promotes proteasomal degradation of bone morphogenetic signaling proteins [κB signaling in osteoblasts has been shown to upregulate Smad7, a general inhibitor of Smad pathway. Finally, a direct inhibitory action of NF-κB on bone formation was demonstrated in vivo to show time- and stage-specific inhibition of IB kinase (IKK) in differentiated osteoblasts, increased trabecular bone mass, and ameliorated ovariectomy-induced bone loss [Many chronic diseases have a local or a systemic inflammatory basis, which has overall deleterious effects on bone mass , 60, leacognized ; recentlin vitro . Tumor nic cells . Pharmaceoblasts . In addione loss .β-catenin signaling and decreased mechanical stimulation with age may contribute to the age-related decline in bone formation. Based on these and other studies, the important role of the Wnt signaling in the control of bone formation has been well recognized as this pathway is suggested to be a potential therapeutic target [Alterations in the Wnt signaling have profound impact on age-related bone loss in mice . Mechanic target . The Wntc target . Nonpharc target .Most drugs currently available for the treatment or prevention of osteoporosis are antiresorptive, including estrogens and selective estrogen receptor modulators, bisphosphonates, and denosumab blocking the RANKL/RANK pathway . It is dCurrently, only strontium ranelate proves to have roles in both the osteoclast inhibition and bone formation promotion. Therefore, further research in the topic may provide insights not only into improving the effects of Sr-containing agents but also into discovering novel and more effective therapies.Several strontium-containing agents, such as Sr fructose, 1,6-diphosphate, and strontium citrate , have beAt the cellular level, it was shown that bone marrow MSC culture, when exposed to strontium (Sr), displayed a significant increase in the expression of the master gene and Runx2, as well as bone sialoprotein (BSP), and this was associated with a significant increase in the formation of colony-forming unit osteoblasts (CFU-obs). Interestingly, the activation of gene expression by Sr varies with the differentiation stage of MSC: Runx2 and BSP in bone marrow MSC; Runx2 and osteocalcin in preosteoblasts; BSP and osteocalcin in mature osteoblasts. Strontium ranelate-treated ovariectomised (OVX) animals exhibited increased bone formation and decreased bone resorption, leading to prevention of trabecular bone loss and improvement of bone microarchitecture and strength –81. ClinIn vitro experiments showed that Sr ranelate had positive effects on osteoblastogenesis and activity of primary rat and human osteoblasts [ in vitro data indicate that strontium promotes the osteogenic differentiation program and reduces osteoblast apoptosis, thereby promoting osteoblastogenesis. The positive effects on preosteoblast eventually result in increased bone nodule formation, a hallmark of in vitro osteogenesis [eoblasts . On the eoblasts , 85 and eoblasts , 87. On eoblasts , 87, 88.ogenesis , 85, 88.Age-related bone loss is generally associated with osteoblast insufficiency relative to the adipogenesis, which is responsible for the progressive adiposity often observed in osteoporosis. The number of mature osteoblasts and adipocytes in bone marrow is influenced by the differentiation of the common mesenchymal progenitor cell towards one phenotype and away from the other. In contrast to the promoting role in osteoblast, strontium exhibits inhibitory role in adipogenesis of MSC . In muriαi and Gαq/11 G-proteins, consequently resulting in decreased intracellular cyclic adenosine monophosphate (cAMP) level, the stimulation of phospholipase Cβ,inositol1,4,5-triphosphate, and the release of intracellular Ca2+ [2+ level, leading to an adjustment in the release of the PTH [2+ because of its similar atomic and ionic properties to Ca2+.The CaSR belongs to subfamily 3 of G-protein coupled receptor family (GPCR) , 91 and lar Ca2+ . The CaS the PTH . SimilarExperimental evidences show that strontium acts on osteoblasts through the CaSR, leading to the activation of MAPK signaling and consequently cell replication. In addition, strontium increases OPG and decreases RANKL expression in osteoblastic cells via the CaSR . HoweverAs mentioned above, strontium also stimulates osteoblast cell growth through the CaSR-independent molecular mechanism. In this regard, a selective inhibitor of FGFR was able to slow down cell growth induced by strontium ranelate in osteoblastic cells, suggesting that the activation of FGFR is a new potential mechanism by which strontium can stimulate osteoblastic cell growth. Activation of FGFR-dependent cell growth is also observed in response to other cations, suggesting that activation of FGFRs may be a new cation-sensing mechanism in osteoblasts .Our own recent work demonstrated that rat sarcoma viral oncogene homolog (RAS), an upstream regulator of MAPK and Akt, was activated by strontium treatment and siRNA-mediated Ras knockdown inhibited strontium-stimulated expression of osteogenic markers . MitogenIt has been confirmed that the effect of strontium on the proliferation and reduced apoptosis of osteoblasts could be neutralized by the selective inhibition of cyclooxygenase-2 (COX-2). The results also indicated that the positive effects of Sr ranelate on osteoblasts depend on the PGE2 production. Strontium induces murine MSCs to express COX-2 that leads to the increased PGE2 production, thus contributing to the increased differentiation of MSC into osteoblasts . This maκ-B ligand (RANKL) [κ-B (RANK) on osteoclast precursors and thereby activates intracellular signaling, resulting in osteoclast differentiation. Osteoblasts can produce OPG, which functions as a decoy receptor for the RANKL and can thereby reduce the production of osteoclasts by inhibiting the differentiation of osteoclast precursors. A positive effect of Sr ranelate was observed on the OPG/RANKL ratio, causing an increased secretion of OPG and a simultaneous reduction of RANKL expression, leading to the suppression of osteoclastogenesis [The primary mechanism by which strontium reduces osteoclast number is to regulate the production of osteoprotegerin (OPG) by osteoblasts and the receptor activator of nuclear factor  (RANKL) , the twoogenesis . The incThe involvement of the calcineurin (Cn)/nuclear factor of activated Tc (NFATc) pathway in bone development and bone remodeling was found in patients who were taking Cn inhibitors such as cyclosporine (CsA) and FK506, developed osteopenia, and had a higher incidence of fractures . It is kβ-catenin into the nucleus through the activation of the CaSR and subsequent activation of Akt-signaling in human osteoblasts [β-catenin transcriptional activity. Inhibitors of the Wnt signaling, DKK1, and sFRP1, a soluble protein with homology to the Wnt-binding site of Frizzled proteins, decreased the Sr-induced expression of osteoblastic genes such as Runx2, ALP, and type 1 collagen [It is reported that strontium promoted the translocation of eoblasts . In addieoblasts , providicollagen . The inhcollagen . Anothercollagen . TherefoA recent study showed that an increase of the insulin-like growth factor (IGF-1) concentration was observed after six-month administration of Sr ranelate at a daily dose of 2.0 g . This reIn summary, multiple mechanisms are involved in strontium effects on bone forming cells. Once exposed to strontium, multiple intracellular signaling pathways and key molecules are activated and orchestrated to promote their survival, proliferation, and differentiation of osteoblasts, which in the meantime produce a series of inflammatory and osteoclast-regulatory factors that may eventually favor bone formation via coupling of both bone resorption and bone formation. in vivo. As the role in stimulating osteoblast proliferation and differentiation becomes well recognized, strontium is widely applied in many bone regeneration biomaterials in various forms, which are briefly presented as below.Tissue engineering is extensively applied in both research and clinical practice of skeletal disease and trauma. For tissue engineering, bioactive materials, with the properties of stimulating osteoblasts and calcimimetics in physiochemical behaviors, are preferential as an implant to facilitate healing or to compensate for bone defects, particularly for osteoporotic fractures where the conventional metallic implant is not applicable because of bone fragility and extremely low bone density. The key of a bioactive material is to form a continuous and highly reactive interface with the surrounding bone tissue to induce abundant bone formation and the ability to be structurally and mechanically compatible with bone tissue and eventually to be replaced by new bones. In some circumstances, it is also demanded that growth factors, drug, and ions are incorporated into such a material, which can in turn be slowly released as the material is biodegradedIn a study by Zhang et al. , strontiThe most important property of bone cement as inorganic filler in load bearing orthopaedic implants is good integration with host bone with reduced bone resorption and increased bone regeneration at the implant interface. Similar to integration into bioglass, strontium has been introduced into bone cement and the mechanical properties, crystallinic properties, and Sr ion release activities have been well evaluated. Sr-containing bone cement has been shown to promote early bone formation by prolonging the release duration of strontium while remaining the strength .2+, CO32−, and Sr2+ for starting materials for the development of nanostructured biodevices for regeneration of osteoporotic bone. Biological-like amounts of Mg and CO3 ions were inserted in the apatite structure to mimic the composition of bone apatite by the addition of Sr ions as antiosteoporotic agent. Based on the hypothesis that the combination of Si and Sr may have synergetic effects on osteoporotic bone regeneration, the porous Sr-substituted calcium silicate (SrCS) ceramic scaffolds combining the functions of Sr and Si elements were developed with the goals to promote osteoporotic bone defect repair [Biomimetic apatites could also be cosubstituted with Sr as well as other elements. In a study by Iafisco et al. , hydroxyt repair . Boron it repair . In addi in vivo biocompatibility and bone healing of the strontium- (Sr-) stabilized bulk glass ceramics for short term implantation of up to 12 weeks in rabbit model and observed excellent healing, which is comparable to that seen during the use of a commercially available implant of HA-based bioglass alone [ in vivo [Implants undergoing early instability or even subsidence correlate with an increased risk of aseptic loosening, subsequently requiring revision. A load-bearing orthopaedic implant therefore needs to possess the properties of well integration with host bone tissue and early fixation by osseointegration of the implant is indeed demanded during surgical practices. This is an even greater challenge during revision replacement surgery using the allograft implant because resorption of the allograft may exceed new bone formation and result in the instability of the prosthesis. Implantation of metal-based joint replacements often results in corrosion and particle release, initiating chronic inflammation leading onto osteoporosis of host bone. A compensative solution is the coating of metal implants with hydroxyapatite (HA) or the use of bulk bioglass. For this purpose, strontium could be incorporated into the material surface or applied in doping surface of an implant. Sabareeswaran et al. therefore tested thess alone . A stronss alone . In a ce in vivo .Membrane materials are particularly useful in guided bone regeneration. In this regard, the effects of a strontium hydroxyapatite- (SrHA-) containing membrane have exhibited higher elasticity and strength than the collagen membrane . MeanwhiStrontium has also been introduced into different hydrogels to be used in bone repair. A study reported that a bone tissue engineering approach in which arginine-glycine-aspartic acid- (RGD-)modified alginate hydrogels are crosslinked with bioactive strontium, zinc, and calcium . It was An amidated carboxymethylcellulose hydrogel enriched with Sr ions was evaluated for its effects of strontium released in the culture medium on osteodifferentiation. It has been shown that strontium released from the gel promotes the osteodifferentiation as shown by the increase of ALP activity, suggesting that the Sr-containing gel could represent a new strategy in bone tissue engineering.Osteoporosis has become a serious and common public health problem. Aging is associated with impaired bone formation as a principal pathogenetic mechanism mediating bone fragility in osteoporosis. At the cellular level, physiology for individual osteoblast survival/growth, apoptosis, migration, and stress response are regulated through elaborated molecular feedback mechanisms. Although further analysis of such mechanisms is still mandatory in order to develop new therapeutics, it is certain that multiple pathways or molecules play important roles from different respects, some of which are emerging as therapeutic targets. Apart from currently proposed or trialed approaches to focusing on agonists/antagonists of certain osteoblastic signaling pathways, such as hPTH1-34 (teriparatide) and antisclerostin or anti-DKK1 antibodies, other strategies aiming at mobilizing skeletal stem cells and activating osteoblastic cell functions are anticipated to be a field worth of much exploring. Strontium, though currently thought as a relatively mild anabolic agent, is considered as a candidate in this respect due to its dual role in regulating osteoblastogenesis/osteoclastogenesis and osteogenesis/adipogenesis via its involvement in multiple pathways. Besides being able to be supplied systemically, the application of strontium is being much extended into various biomaterials and thereby tissue engineering strategies for the local bone lesions and defects.Further, on the mounting evidences and rapid progress in revealing key molecular factors and signaling pathways regulating bone forming cells and osteoporotic conditions, a systems biology approach to coherently relating these factors with mathematical modeling would be particularly helpful to better understand and evaluate therapeutics. Future progress in this field will hopefully provide opportunities for exploring drug discovery.


Plant organelles produce retrograde signals to alter nuclear gene expression in order to coordinate their biogenesis, maintain homeostasis, or optimize their performance under adverse conditions. Many signals of different chemical nature have been described in the past decades, including chlorophyll intermediates, reactive oxygen species (ROS), and adenosine derivatives. While the effects of retrograde signaling on gene expression are well understood, the initiation and transport of the signals and their mode of action have either not been resolved, or are a matter of speculation. Moreover, retrograde signaling should be considered as part of a broader cellular network, instead of as separate pathways, required to adjust to changing physiologically relevant conditions. Here we summarize current plastid retrograde signaling models in plants, with a focus on new signaling pathways, SAL1-PAP, methylerythritol cyclodiphosphate (MEcPP), and β-cyclocitral (β-CC), and outline missing links or future areas of research that we believe need to be addressed to have a better understanding of plant intracellular signaling networks. Chloroplasts originated from free-living cyanobacteria that were engulfed by the eukaryotic cell ancestor  and singlet oxygen (1O−2) by excess oxidative power is a third mechanism that can trigger specific changes in nuclear gene expression   mutants  genes such as Light Harvesting Complex b (LHCB) during perturbations of chloroplast development by NF.Classical retrograde signals in plants generally involved artificially stressing the plant cells by treating the plants with the herbicide norflurazon (NF), which is an inhibitor of carotenoid biosynthesis that can perturb chloroplast development  gene in the gain-of-function gun1-6D mutant. FC1 over expression leads to the accumulation of PhANGs in the presence of NF . This response seems to be specific to the activity of FC1, as over expression of the other chloroplast-localized FC2 did not increase PhANG expression. Unexpectedly, and unlike Mg-ProtoIX, a reduction in total haem, rather than accumulation, occurs in the FC1 OX, and wild type plants after NF treatment   Despite a paucity of discovery of retrograde signals in the last decade, several signaling pathways have been recently proposed in the past 12 months. These novel signals include products of secondary metabolism  and Early Light Induced Protein 2 (ELIP2), altered metabolome and 50% increased survival under water limiting conditions  is a novel metabolite discovered to play a role as a plastid signal during drought and HL stress in Arabidopsis  is unknown and merits further research.PAP most likely regulates gene expression by altering RNA metabolism mediated by 5–3′ exoribonucleases (XRNs). There are three XRNs in Arabidopsis; two encode the nuclear-localized XRN2 and XRN3 and are homologues of yeast Xrn2p/Rat1p. XRN2 and XRN3 act on uncapped RNAs like the excised hairpin loops in precursor miRNA transcripts  accumulates MEcPP and HPL transcript, with up-regulation of salicylic acid (SA) production, and that of the ICS1 (encoding a SA-biosynthetic enzyme) transcript. This results in ceh1 mutants being more resistant to infection by Pseudomonas syringae, a biotrophic pathogen. Targeted silencing of all the enzymes in the MEP pathway indicates that only the accumulation of MEcPP is responsible for SA accumulation and induction of HPL. Moreover, feeding experiments demonstrates that MEcPP can directly regulate HPL. Thus, it seems that unlike the mechanism mediated by chlorophyll precursors where the flux through the pathway is important for retrograde signaling  identified the isoprenoid precursor methylerythritol cyclodiphosphate (MEcPP) as a potential signal from the chloroplast , accumulates more than 1.5-fold after 1 h of stress treatment, which is accompanied by the induction of 1O2 stress-responsive genes  that translocate to the nucleus and alter gene expression under specific conditions have been recently described. Arguably, metabolite signals that could be modulated by specific proteins better fit in the “signal” category Leister, , but idePHD type transcription factor with transmembrane domains) is involved in retrograde signal pathways that regulate PhANGs expression under different types of stress conditions  similar to that of gun1, and abi4 during HL treatment. The involvement of PTM in the same pathway as GUN1 and ABI4 was confirmed by analyses of the corresponding double mutants. Interestingly, the levels of H3K4m3 methylation in the ABI4 promoter increased with the same stress treatments activating the PTM-dependent transcription of the ABI4 gene. An unexpected observation was the lack of constitutive repression of LHCB in the absence of NF in plant expressing soluble PTM. Although this may indicate that additional GUN1 mediated signals may be required, this work demonstrates that PTM mediates several retrograde signaling pathways.The chloroplast envelope-bound PTM (Another mobile protein that could potentially be involved in retrograde signaling is Whirly1 protein (Isemer et al., It is relatively easy to envision a linear type of mechanism whereby the available pool of signal interacts with target proteins to regulate gene expression (Estavillo et al., Unlike the case of photosynthetic derived signals, it is less clear how the different triggers are sensed in most retrograde signaling pathways. For example, how is the protease mediated cleavage of PTM regulated by HL (Sun et al., The movement of the signal between organelles is a key feature of a metabolite, or protein capable of conveying information into the nucleus. Passive diffusion and active transport are the two most likely scenarios depending on the type of signal. However, monitoring this movement can be technically challenging. Analytical methods for metabolic profiling, such as cell fractionation in conjunction with non-aqueous fractionation, GC/MS- and LC/MS-based, and HPLC, or confocal microscopy for specific signals can be attempted. For instance, the observation that Mg-ProtoIX can accumulate in both chloroplast and cytosol suggests that this metabolite can be exported from site of origin, which may result in the repression of photosynthetic genes under stress (Ankele et al., sal1 mutants, supporting the hypothesis that PAP moves in between organelles (Estavillo et al., An alternative to direct measurements of the signal is the use of genetic approaches. Genetically encoded biosensors specific for putative signals, such as those developed for sugars and hormones (Frommer et al., in vitro. It still remains unclear whether this transporter plays any role in the PAP signaling pathway, especially during abiotic stress. Modulation of the transporter activity could be a major point of flux control, as also considered elsewhere (Leister, Identification of transporters that regulate the flux of the signal from the plastid into the cytosol is critical for understanding a signal transduction pathway. Although some of the proposed signals, like β-CC, could freely diffuse from the site of origin (Ramel et al., Leister, .Affinity chromatography against putative signals coupled to proteomic analyses is a logical approach to dissect the components of the retrograde signaling pathways. This strategy rendered a large number of proteins associated to oxidative stress that bound to Mg-ProtoIX covalently linked gel matrix (Kindgren et al., Some of the retrograde mechanisms have been identified using inducible reporter genes, a common strategy for dissecting signaling pathways (Susek et al., alx8 mutant do not seem to be affected by XRNs (Estavillo et al., Although the general view of retrograde metabolic signals is that of the “classical” model, where one signal acts through a rather linear pathway, it is likely that a “one signal, many targets” scenario is more common than anticipated, especially when considering metabolites that could bind to proteins. For example, 60% of genes misregulated in the SAL1 mutant There are several recent examples of some classical retrograde signals converging with other networks such as light signaling (Ruckle and Larkin, Several new metabolite retrograde signals have been recently proposed (Estavillo et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.