The relevance of compartmentation for cysteine synthesis in phototrophic organisms

In the vascular plant Arabidopsis thaliana, synthesis of cysteine and its precursors O-acetylserine and sulfide is distributed between the cytosol, chloroplasts, and mitochondria. This compartmentation contributes to regulation of cysteine synthesis. In contrast to Arabidopsis, cysteine synthesis is exclusively restricted to chloroplasts in the unicellular green alga Chlamydomonas reinhardtii. Thus, the question arises, whether specification of compartmentation was driven by multicellularity and specified organs and tissues. The moss Physcomitrella patens colonizes land but is still characterized by a simple morphology compared to vascular plants. It was therefore used as model organism to study evolution of compartmented cysteine synthesis. The presence of O-acetylserine(thiol)lyase (OAS-TL) proteins, which catalyze the final step of cysteine synthesis, in different compartments was applied as criterion. Purification and characterization of native OAS-TL proteins demonstrated the presence of five OAS-TL protein species encoded by two genes in Physcomitrella. At least one of the gene products is dual targeted to plastids and cytosol, as shown by combination of GFP fusion localization studies, purification of chloroplasts, and identification of N termini from native proteins. The bulk of OAS-TL protein is targeted to plastids, whereas there is no evidence for a mitochondrial OAS-TL isoform and only a minor part of OAS-TL protein is localized in the cytosol. This demonstrates that subcellular diversification of cysteine synthesis is already initialized in Physcomitrella but appears to gain relevance later during evolution of vascular plants.     

Metabolic alteration of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension cultures overexpressing <Emphasis Type="Italic">geraniol synthase</Emphasis> in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.     

Ancestral and more recently acquired syntenic relationships of MADS-box genes uncovered by the <Emphasis Type="Italic">Physcomitrella patens</Emphasis> pseudochromosomal genome assembly

Key message

The Physcomitrella pseudochromosomal genome assembly revealed previously invisible synteny enabling realisation of the full potential of shared synteny as a tool for probing evolution of this plant’s MADS-box gene family.

Abstract

Assembly of the sequenced genome of Physcomitrella patens into 27 mega-scaffolds (pseudochromosomes) has confirmed the major predictions of our earlier model of expansion of the MADS-box gene family in the Physcomitrella lineage. Additionally, microsynteny has been conserved in the immediate vicinity of some recent duplicates of MADS-box genes. However, comparison of non-syntenic MIKC MADS-box genes and neighbouring genes indicates that chromosomal rearrangements and/or sequence degeneration have destroyed shared synteny over longer distances (macrosynteny) around MADS-box genes despite subsets comprising two or three MIKC genes having remained syntenic. In contrast, half of the type I MADS-box genes have been transposed creating new syntenic relations with MIKC genes. This implies that conservation of ancient ancestral synteny of MIKC genes and of more recently acquired synteny of type I and MIKC genes may be selectively advantageous. Our revised model predicts the birth rate of MIKC genes in Physcomitrella is higher than that of type I genes. However, this difference is attributable to an early tandem duplication and an early segmental duplication of MIKC genes prior to the two polyploidisations that account for most of the expansion of the MADS-box gene family in Physcomitrella. Furthermore, this early segmental duplication spawned two chromosomal lineages: one with a MIKC ^C gene, belonging to the PPM2 clade, in close proximity to one or a pair of MIKC* genes and another with a MIKC ^C gene, belonging to the PpMADS-S clade, characterised by greater separation from syntenic MIKC* genes. Our model has evolutionary implications for the Physcomitrella karyotype.

     

Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.     

A tomato plastidic ATP/ADP transporter gene <Emphasis Type="Italic">SlAATP</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

A plastidic ATP/ADP transporter (AATP) is responsible for importing ATP from the cytosol into plastids. Increasing the ATP supply is a potential way to facilitate anabolic synthesis in heterotrophic plastids of plants. In this work, a gene encoding the AATP protein, named SlAATP, was successfully isolated from tomato. Expression of SlAATP was induced by exogenous sucrose treatment in tomato. The coding region of SlAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of SlAATP significantly increased the starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of StAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AtAGPase), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild-type (WT). These findings suggest that SlAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of SlAATP expression might be used for increasing starch accumulation of plants in the future.     

Signaling in the plant cytosol: cysteine or sulfide?

Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel l-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance.     

Successful Fertilization Requires the Presence of at Least One Major O-Acetylserine(thiol)lyase for Cysteine Synthesis in Pollen of Arabidopsis

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.Sulfur assimilation in plants is hallmarked by two reaction sequences, namely sulfate reduction and Cys synthesis. The sulfate reduction pathway consists of three steps and produces sulfide from sulfate, which is available in the soil and transported into the roots by specific transporters (Takahashi et al., 2011). Sulfide is subsequently incorporated into the amino acid O-acetylserine (OAS) by O-acetylserine(thiol)lyase (OAS-TL; EC 2.5.1.47) to produce Cys (Hell and Wirtz, 2011). Cys then serves as the sulfur source for all organic metabolites containing reduced sulfur in plants, including proteins, cofactors, and secondary metabolites. The tripeptide glutathione (GSH) is one of the most important Cys-derived metabolites, since it has an important function in redox homeostasis and the control of development (Meyer and Rausch, 2008). Impaired GSH synthesis negatively affects growth of the shoot and root system of Arabidopsis (Arabidopsis thaliana; Vernoux et al., 2000; Xiang et al., 2001), and loss-of-function mutants for the first enzyme (GSH1, Glu-Cys ligase; EC 6.3.2.2) or the second enzyme (GSH2, glutathione synthase; EC 6.3.2.3) of the two-step pathway leading to GSH formation show an embryo- and seedling-lethal phenotype, respectively (Cairns et al., 2006; Pasternak et al., 2008).Cys synthesis by OAS-TL constitutes the direct link between carbon and nitrogen (OAS) as well as sulfur (sulfide) metabolism and, therefore, can be designated as one of the central reactions in plant primary metabolism. The genome of the model plant Arabidopsis encodes nine OAS-TL-like enzymes: OAS-TL A1 (At4g14880), OAS-TL B (At2g43750), and OAS-TL C (At3g59760) are the major isoforms and are localized in the cytosol, plastids, and mitochondria, respectively (Jost et al., 2000). OAS-TL A2 (At3g22460) encodes a truncated and nonfunctional protein (Jost et al., 2000). In the following, therefore, OAS-TL A1 is referred to as OAS-TL A. CYS D1 (At3g04940) and CYS D2 (At5g28020) show OAS-TL activity in vitro (Yamaguchi et al., 2000). Whether they contribute to net Cys synthesis in vivo is unknown (Heeg et al., 2008). CS26 (At3g03630) encodes a plastidic S-sulfocysteine synthase, which prefers thiosulfate instead of sulfide as substrate and produces S-sulfocysteine (Bermúdez et al., 2010). Whether thiosulfate is taken up from the soil or formed within the plant is unclear, but its presence in Arabidopsis was demonstrated (Tsakraklides et al., 2002). However, the synthesis of S-sulfocysteine from thiosulfate potentially constitutes an alternative sulfur fixation pathway. So far, CS26 was shown to be important for the regulation of redox homeostasis in plastids under certain stress conditions (Bermúdez et al., 2010). DES1 (At5g28030; formerly known as CS-LIKE) is a Cys desulfhydrase (EC 4.4.1.15) that releases sulfide in the cytosol (Alvarez et al., 2010). As a Cys-consuming enzyme, it contributes to Cys homeostasis, especially in late vegetative development and under certain stress conditions (Alvarez et al., 2010, 2012). CYS C1 (At3g61440), finally, encodes a mitochondrial β-cyanoalanine synthase (EC 4.4.1.9), which detoxifies cyanide by incorporation into Cys (Yamaguchi et al., 2000; Watanabe et al., 2008a; García et al., 2010). The major isoforms OAS-TL A, OAS-TL B, and OAS-TL C as well as CYS D1 and CYS D2 can interact with serine acetyltransferase (SAT; EC 2.3.1.30) in the cysteine synthase complex (CSC; Heeg et al., 2008). Although SAT acetylates Ser at the hydroxyl group to form OAS, the direct substrate of OAS-TL, formation of the CSC has no substrate-channeling function but contributes to the demand-driven regulation of Cys synthesis (Hell and Wirtz, 2011).The subcellular compartmentation of Cys precursor formation is a remarkable feature of Cys synthesis in higher plants that implies a high degree of regulation between the participating compartments: while sulfate is exclusively reduced to sulfide in plastids (Takahashi et al., 2011), the synthesis of OAS and the incorporation of sulfide take place in all three compartments where SAT and OAS-TL are present, namely in the cytosol, plastids, and mitochondria. Reverse genetics approaches proved a certain redundancy between the different SAT and OAS-TL isoforms, which demonstrates that sulfide, OAS, and Cys can be exchanged between these compartments (Haas et al., 2008; Heeg et al., 2008; Watanabe et al., 2008a, 2008b). Indeed, sulfide can easily diffuse through membranes (Mathai et al., 2009), but OAS and Cys need to be actively transported. However, the identity of these transporters is unknown. Although sulfide, OAS, and Cys can pass the mitochondrial membrane (Wirtz et al., 2012), the loss-of-function mutant for mitochondrial OAS-TL C is the only single oastl knockout mutant that displays a significant growth phenotype (Heeg et al., 2008). This result was astonishing, since OAS-TL C contributes only 5% to extractable foliar OAS-TL activity (Heeg et al., 2008). The retarded growth of the oastlC mutant, however, cannot be explained by the lack of sulfide detoxification in mitochondria by OAS-TL C, due to an alternative detoxification mechanism for sulfide in mitochondria (Birke et al., 2012). These data question the total redundancy between the different OAS-TL isoforms and suggest specific functions in the different subcellular compartments.Despite its central position in the primary metabolism of higher plants, fundamental questions about Cys synthesis are still unanswered. First, the contribution of OAS-TL-like proteins, especially CYS D1, CYS D2, and CS26, to the fixation of sulfur in planta is unknown. Second, the significance of Cys synthesis by the major OAS-TL proteins in the different subcellular compartments during sporophyte and gametophyte development is unclear. In this study, we addressed these questions using a reverse genetics approach. We were able to prove that fixation of sulfur is carried out exclusively by the major OAS-TL isoforms OAS-TL A, OAS-TL B, and OAS-TL C and elucidated specific functions for OAS-TL A in the cytosol and OAS-TL C in mitochondria of leaf cells. Furthermore, we demonstrate that Cys can be supplied by the mother plant for the development of female gametophytes lacking OAS-TL activity. In contrast, the presence of at least one functional OAS-TL isoform is essential in the male gametophyte.     

Lamelloplasts and minichloroplasts in Begoniaceae: iridescence and photosynthetic functioning

Iridoplasts (modified plastids in adaxial epidermal cells) reported from Begonia were originally hypothesized to cause iridescence, which was broadly accepted for decades. However, several species of Begonia with iridoplasts are not iridescent causing confusion. Here chloroplast ultrastructure was observed in 40 taxa of Begoniaceae to explore the phenomenon of iridescence. However, 22 Begonias and Hillebrandia were found to have iridoplasts, but only nine display visually iridescent blue to blue-green leaves. Unexpectedly, a new type of plastid, a ‘minichloroplast,’ was found in the abaxial epidermal cells of all taxa, but was present in adaxial epidermal cells only if iridoplasts were absent. Comparative ultrastructural study of iridoplasts and a shading experiment of selected taxa show that a taxon with iridoplasts does not inevitably have visual iridescence, but iridescence is greatly affected by the spacing between thylakoid lamellae (stoma spacing). Thus, we propose instead the name ‘lamelloplast’ for plastids filled entirely with regular lamellae to avoid prejudging their function. To evaluate photosynthetic performance, chlorophyll fluorescence (F _v /F _m ) was measured separately from the chloroplasts in the adaxial epidermis and lower leaf tissues by using leaf dermal peels. Lamelloplasts and minichloroplasts have much lower photosynthetic efficiency than mesophyll chloroplasts. Nevertheless, photosynthetic proteins (psbA protein of PSII, RuBisCo and ATPase) were detected in both plastids as well as mesophyll chloroplasts in an immunogold labeling. Spectrometry revealed additional blue to blue-green peaks in visually iridescent leaves. Micro-spectrometry detected a blue peak from single blue spots in adaxial epidermal cells confirming that the color is derived from lamelloplasts. Presence of lamelloplasts or minichloroplasts is species specific and exclusive. High prevalence of lamelloplasts in Begoniaceae, including the basal clade Hillebrandia, highlights a unique evolutionary development. These new findings clarify the association between iridescence and lamelloplasts, and with implications for new directions in the study of plastid morphogenesis.     

A soybean plastidic ATP/ADP transporter gene, <Emphasis Type="Italic">GmAATP</Emphasis>, is involved in carbohydrate metabolism in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.     

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified.     

Nuclear genome sequence of the plastid-lacking cryptomonad <Emphasis Type="Italic">Goniomonas avonlea</Emphasis> provides insights into the evolution of secondary plastids

Background

The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea—the first for any goniomonad—to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily.

Results

We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~?92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida.

Conclusion

We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic “rewiring” that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

     

Gyrfalcons <Emphasis Type="Italic">Falco rusticolus</Emphasis> adjust <Emphasis Type="Italic">CTNS</Emphasis> expression to food abundance: a possible contribution to cysteine homeostasis

Melanins form the basis of animal pigmentation. When the sulphurated form of melanin, termed pheomelanin, is synthesized, the sulfhydryl group of cysteine is incorporated to the pigment structure. This may constrain physiological performance because it consumes the most important intracellular antioxidant (i.e., glutathione, GSH), of which cysteine is a constitutive amino acid. However, this may also help avoid excess cysteine, which is toxic. Pheomelanin synthesis is regulated by several genes, some of them exerting this regulation by controlling the transport of cysteine in melanocytes. We investigated the possibility that these genes are epigenetically labile regarding protein intake and thus contribute to cysteine homeostasis. We found in the Icelandic population of gyrfalcon Falco rusticolus, a species that pigments its plumage with pheomelanin, that the expression of a gene regulating the export of cystine out of melanosomes (CTNS) in feather melanocytes of developing nestlings increases with food abundance in the breeding territories where they were reared. The expression of other genes regulating pheomelanin synthesis by different mechanisms of influence on cysteine availability (Slc7a11 and Slc45a2) or by other processes (MC1R and AGRP) was not affected by food abundance. As the gyrfalcon is a strict carnivore and variation in food abundance mainly reflects variation in protein intake, we suggest that epigenetic lability in CTNS has evolved in some species because of its potential benefits contributing to cysteine homeostasis. Potential applications of our results should now be investigated in the context of renal failure and other disorders associated with cystinosis caused by CTNS dysfunction.     

Salinity induced changes in the chloroplast proteome of the aquatic pteridophyte <Emphasis Type="Italic">Azolla microphylla</Emphasis>

The growth of the nitrogen fixing aquatic pteridophyte Azolla microphylla is severely affected by salinity. Salinity exposure (0.5%) resulted in significant reduction in chlorophyll a and b content, altered chl a/b ratio and photosynthetic efficiency (Fv/Fm). Chloroplasts maintain photosynthesis but are highly sensitive to salinity stress. Chloroplast proteins extracted from A. microphylla was separated by two-dimensional electrophoresis (2DE) and approximately 200 proteins were observed on each gel. Forty two differentially expressed protein spots were detected and out of this 17 could be identified through MALDI-TOF-MS/MS analysis. Out of the 17 identified proteins, 15 were found to be down regulated and 2 proteins were up regulated. Most of the down regulated proteins were associated with Calvin cycle, ATP synthesis, oxygen evolution, photosystem I and ROS scavenging. The results show changes in proteome dynamics of the chloroplasts of A. microphylla and such changes may lead to reduction in growth and metabolism. The primary target of salinity in A. microphylla is photosynthesis and the changes in the proteome dynamics of the chloroplasts lead to reduced growth.     

Coding single nucleotide polymorphisms and SmCPS1 and SmKSL1 subcellular localization are associated with tanshinone biosynthesis in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bunge roots

Tanshinone is one of the major medicinal components of the roots of Salvia miltiorrhiza Bunge, and SmCPS1 and SmKSL1 are key enzymes in the tanshinone biosynthesis pathway. To increase our understanding of the coding single nucleotide polymorphisms (cSNPs) involved in tanshinone biosynthesis, seven S. miltiorrhiza landraces were examined. Our results revealed that the tanshinone content was significantly different among the seven landraces. In total, 48 cSNPs in SmCPS1 and 47 cSNPs in SmKSL1 were identified, and of these, 38 and 42 cSNPs, respectively, were associated with tanshinone content. The highest A/G and C/T base substitution rates were in SmCPS1 and SmKSL1, respectively. SmKSL1 expression was significantly, positively correlated with tanshinone IIA and tanshinone I contents, and SmCPS1 expression was significantly associated with tanshinone IIA content. Interestingly, subcellular SmCPS1 and SmKSL1 expression was enriched in the plastids. Therefore, cSNPs of SmCPS1 and SmKSL1 are involved in tanshinone biosynthesis in the plastids, where SmCPS1 and SmKSL1 enzymes catalyze tanshinone production in this species.     

Effect of Cytokinin and Auxin Treatments on Morphogenesis,Terpenoid Biosynthesis,Photosystem Structural Organization,and Endogenous Isoprenoid Cytokinin Profile in <Emphasis Type="Italic">Artemisia alba</Emphasis> Turra In Vitro

Developmental pattern modification in essential oil bearing Artemisia alba Turra was obtained by exogenous plant growth regulator (PGRs) treatments in vitro. Enhanced rooting (in PGR-free and auxin-treated plants) led to elevation of the monoterpenoid/sesquiterpenoid ratio in the essential oils of aerials. On the contrary, root inhibition and intensive callusogenesis [combined cytokinin (CK) and auxin treatments] reduced this ratio more than twice, significantly enhancing sesquiterpenoid production. Both morphogenic types displayed sesquiterpenoid domination in the underground tissues, which however differed qualitatively from the sesquiterpenoids of the aerials, excluding the hypothesis of their shoot-to-root translocation and implying the possible role of another signaling factor, affecting terpenoid biosynthesis. Inhibited rooting also resulted in a significant drop of endogenous isoprenoid CK bioactive-free bases and ribosides as well as CK N-glycoconjugates and in decreased trans-zeatin (transZ):cis-zeatin (cisZ) ratio in the aerials. Marked impairment of the structural organization of the photosynthetic apparatus and chloroplast architecture were also observed in samples with suppressed rooting. It is well known that in the plant cell monoterpenoid and transZ-type CKs biogenesis are spatially bound to plastids, while sesquiterpenoid and cisZ production are compartmented in the cytosol. In the present work, interplay between the biosynthesis of terpenoids and CK bioactive free bases and ribosides in A. alba in vitro via possible moderation of chloroplast structure has been hypothesized.     

Phylogenetic analysis of proteins involved in the stringent response in plant cells

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ppx families indicated that plant gppA/ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol.