Low abundance does not mean less importance in cysteine metabolism

The cysteine molecule plays an essential role in cells because it is part of proteins and because it functions as a reduced sulfur donor molecule. In addition, the cysteine molecule may also play a role in the redox signaling of different stress processes. Even though the synthesis of cysteine by the most abundant of the isoforms of O-acetylserine(thiol) lyase in the chloroplast, the mitochondria and the cytosol is relatively well-understood, the role of the other less common isoforms homologous to O-acetylserine(thiol)lyase is unknown. Several studies on two of these isoforms, one located in the cytosol and the other one in the chloroplast, have shown that while one isoform operates with a desulfhydrase activity and is essential to regulate the homeostasis of cysteine in the cytosol, the other, located in the chloroplast, synthesizes S-sulfocysteine. This metabolite appears to be essential for the redox regulation of the chloroplast under certain lighting conditions.Key words: cysteine, S-sulfocysteine, desulfhydrase, sulfur metabolism, redox regulation, ArabidopsisCysteine occupies a central position in the plant primary and secondary metabolism due to its biochemical functions. Cysteine is the first organic compound with reduced sulfur synthesized by the plant in the photosynthetic primary sulfate assimilation. The importance of cysteine for plants derives from its role as an amino acid in proteins but also because of its functions as a precursor for a huge number of essential bio-molecules, such as many plant defense compounds formed in response to different environmental adverse conditions.^1,2 All of these bio-molecules contain sulfur moieties that act as functional groups and are derived from cysteine, and therefore, are intimately linked via their biosynthetic pathways.In addition to the final destination of the reduced sulfur atom in the primary and secondary metabolism of cells, the thiol residue of the cysteine molecule is a functional group that translates the physico-chemical signal (redox) of ROS and RNS into a functional signal, altering the properties of small molecules such as GSH or proteins whose enzymatic or functional properties depend on the redox state of its cysteine residues.³Sulfate is the major sulfur form available to plants. Sulfate is taken up to plant cells through specific sulfate transporters and is activated to adenosine 5′-phosphosulfate (APS). The reduction of the activated sulfate form, APS, is linked to plastids and the photosynthetic activity; therefore, APS is reduced to sulfite by the APS reductase using two GSH molecules as donors of the two electrons required in this step. Sulfite is further reduced to sulfide by the sulfite reductase that uses photosynthetically reduced ferredoxine (Fd) as an electron donor of the six required electrons. The biosynthesis of cysteine is further accomplished by the sequential reaction of two enzymes: First, the serine acetyltransferase (SAT) synthesizes the intermediary product, O-acetylserine (OAS), from acetyl-CoA and serine; and second, the O-acetylserine(thiol)lyase (OASTL) incorporates the sulfide to OAS producing the cysteine. Recently, much progress has been made toward understanding the action of the O-acetylserine(thiol)lyase (OASTL) enzyme, one of the enzymes responsible for the biosynthesis of cysteine, using as a model system the plant Arabidopsis thaliana. The focus of the research has been mainly placed on the most abundant enzymes based on their involvement in the primary sulfate assimilation pathway. Biochemical and molecular analysis of the major OASTL knockout mutants in Arabidopsis thaliana revealed that part of the produced sulfide is incorporated to O-acetylserine to form cysteine in the chloroplast with the assistance of the OAS-B isoform. However, most of the chloroplastic sulfide overflows and escapes into the cytosol and the mitochondria, where it is also assimilated into cysteine by the OAS-A1 and OAS-C isoforms, respectively.^4–6The three major OASTL isoforms seem to be redundant under normal growth conditions. However, our investigations on the major cytosolic isoform, the OAS-A1, revealed new insights on the function of this enzyme as a determinant of the antioxidative capacity of the cytosol.⁷ The OASTL homolog, CYS-C1, exhibits OASTL activity, but in fact, it is a β-cyanoalanine synthase enzyme that uses cysteine to detoxify cyanide within the mitochondria.⁸ Furthermore, Arabidopsis cells contain four additional O-acetylserine(thiol)lyase isoforms encoded by the CYS-D1 (At3g04940), CYS-D2 (At5g28020), CS26 (At3g03630) and CS-LIKE (At5g28030) genes with unknown function. Are these four isoforms authentic OASTL and are, therefore, redundant enzymes or do they have different activities and, therefore, different functions?Our recent research on the less-common isoforms, CS-like and CS26, shed light on this issue, and we are decoding two important aspects of the sulfur metabolism in plants.^9,10 The CS-LIKE protein was identified by sequence homology upon the completion of the sequencing of the Arabidopsis genome. Because of its cytosolic localization, it is thought to have an auxiliary function with respect to the major cytosolic isoform, the OAS-A1. The characterization of the purified recombinant protein has shown that the CS-LIKE isoform catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate; thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and it is designated as DES1 (Fig. 1). This enzyme is important for maintaining the homeostasis of cysteine in the cell, and the loss of function of this protein in knockout mutant plants results in higher levels of cysteine and glutathione. This increased level of soluble thiols results also in a higher antioxidant capacity of the plant, which, in turn, becomes more resistant to abiotic stress phenomena such as the presence of heavy metals or hydrogen peroxide. This observation may indicate that the regulation of this enzyme may be a key component of the plant physiological processes that involve redox reactions. Cytosolic cysteine degrading enzymes with desulfhydrase activity has been found in plants, but the protein responsible for this activity remained unisolated until now that it is revealed with our investigation on DES1.¹¹ From the standpoint of biotechnology, plants with this modified enzyme may result in abiotic stress-resistant lines that deserve to be studied.Open in a separate windowFigure 1Biosynthesis of cysteine and S-sulfocysteine in the chloroplast and cytosol of Arabidopsis and subcellular localization of the responsible enzymes. The cytosolic and plastidial O-acetylserine(thiol)lyase, L-cysteine desulfhydrase and S-sulfocysteine synthase are shown in red. A single representative of a grana thylakoid is shown as a grey oval compartment.The other less common enzyme studied, called CS26 and localized in the chloroplast, has proved to be an enzyme with S-sulfocysteine synthase activity.¹⁰ This enzyme synthesizes the incorporation of thiosulfate to O-acetylserine to form S-sulfocysteine (RSSO₃⁻). This activity, discovered for the first time in plants, was previously reported in bacteria where the biosynthesis of cysteine can be accomplished by two enzymes encoded by the cysK and cysM genes.^12,13 This enzyme activity is essential for the chloroplast function under long-day growing conditions but seems to be superfluous under short-day conditions. Morphologic and biochemical phenotype comparisons of the knockout oas-b and cs26 highlight the importance of the metabolite S-sulfocysteine and not the cysteine in the redox control of the chloroplast. Under long-day growth conditions, the cs26 mutants exhibit a reduction in size and show leaf paleness, have reductions in the chlorophyll content and photosynthetic activity, and are not able to properly detoxify reactive oxygen species, which are accumulated to high levels. None of these changes are observed in the oas-b mutant.Although we do not know the function of the S-sulfocysteine molecule in the chloroplast, two aspects are important to note. On the one hand, the enzyme CS26 can be located in the chloroplast''s lumen in opposition to the enzyme OAS-B, which is located in the stroma. The second aspect is the difference in chemical reactivity of S-sulfocysteine and cysteine. The S-sulfocysteine has two sulfur atoms with different degrees of oxidation, −1 and +5; therefore, it may act as an oxidant molecule by reacting with reduced thiols forming a disulfide bridge and releasing sulfite.¹⁴ We have suggested that a putative target of S-sulfocysteine can be the STN7 kinase, which contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved cysteines that are critical for its activity. A disulfide bridge between these two cysteines is required for the kinase activity, but how the redox states of these two cysteines are regulated in the lumen remains an open question.¹⁵ In general, how the thiol oxidation of proteins located in the thylakoid lumen takes place is still unclear because no sulfhydryl oxidases have been identified in this compartment. In fact, this process is highly important because the chaperones and peptidyl-prolyl cis-trans isomerases, such as the AtFKBP13, need to be oxidized in order to be functional in the lumen and to regulate the folding of the Rieske protein.^16–18     

Thioredoxin and Redox Control within the New Concept of Oxidative Signaling

During the last decade, plant thioredoxins (TRX) h-type have been shown to be implicated in several new roles like the protection against the oxidative stress by their ability to reduce some antioxidant proteins as peroxiredoxins (PRX) or methionine-sulphoxide-reductases (MSR). However, the concept of the oxidative stress is changing and this fact raises the question of the TRX roles in this new context. In the January issue of Plant Physiology, we have presented two TRXsh from Pisum sativum differently involved in the control of the redox status. PsTRXh1 is an h-type TRX that acts by reducing classical antioxidant proteins. PsTRXh2 seems to be also involved in redox control, however it could act contrary to its counterpart h1. Both proteins may play antagonistic roles in pea in order to lead a better control of the redox status.Key Words: abiotic stress, oxidative signalling, thioredoxins, Pisum sativum, ROSHigh concentration of reactive oxygen species (ROS) in plant cells involve the activation of different antioxidant systems which reestablish the redox status leading to better physiological conditions. On the other hand, it has been well established that at certain levels, the ROS act as second messengers in signal transduction cascades in several processes in plant cells.¹ At the light of these events, it has been proposed the reevaluation of the concept of oxidative stress towards “oxidative signalling.”² This concept involves all the cellular mechanisms that let the plant cells sense and act in response to modified environmental conditions. Several cellular systems are involved in such role, and in these last years, plant TRXs have been shown to be involved in several number of metabolic pathways linked to the regulation of the redox imbalance,³ mainly for the case of the h-type cluster of the TRXs.^4–6In our last work,⁷ we have described two pea TRXs of the h-type cluster, PsTRXh1 and PsTRXh2 that are differentially, and even antagonistically, involved in the redoxregulation control, probably through their interaction with different target proteins. We proposed that PsTRXh1 might be involved in the control of the ROS levels in pea tissues due to its ability to interact with several antioxidant proteins in vivo. It is now very well known that some members of the TRX family reduce PRX,^8–10 an antioxidant enzyme involves in the direct deactivation of some oxidant agents or the MSR,^11,14,15 in charge of the recovery of the oxidized methionine, both in a very specific manner. Due to the increase of PsTRXh1 both at gene expression and protein levels in plant heterotrophic tissues in response to the H₂O₂ treatment, and because it is also capable of conferring resistance towards hydrogen peroxide when produced in a yeast trx1Δ trx2Δ strain,¹⁶ one function of this TRX member could be the reduction in vivo of some PRX and/or MSRA counterparts in Pea tissues in the context of the oxidative signalling.Interestingly, PsTRXh2 gene and its corresponding protein showed very different behaviours to that presented by its homologous h1, reinforcing the idea that some TRX isoforms in plants are capable of functional specificity in vivo. PsTRXh2 is expressed in all tissues assays, mainly in roots, but at an extremely low level compared with that of PsTRXh1. Its divergent functional behaviour was confirmed both in Pea plantlets and yeast. In fact, contrary to PsTRXh1, PsTRXh2 provides hypersensitivity in the yeast trx1Δ trx2Δ mutant. We explained the different behaviour by suggesting that PsTRXh2 might interact with some target(s) involved either directly or indirectly in hydrogen peroxide detoxification, either by compromising the target function in resistance to the ROS or by reinforcing the target function in producing sensitivity to H₂O₂. Most probably, PsTRXh1 and PsTRXh2 interact with very different partners, and the characterization of such targets may help in the deciphering of PsTRX isoforms. As short-term future experiments, using the TRX-specific two-hybrid system that was published recently,⁸ comparative efficiencies of PsTRX isoforms could be performed to reduce some putative target involved in H₂O₂ detoxification, including PsTRXh3 and PsTRXh4.¹⁷ Unravelling Pea TRX interactome should help in deciphering the function of each isoform.However, we have also considered the possibility that PsTRXh2 could interact with the same target that PsTRXh1, but producing an opposite effect. In the yeast context, the protein targeted by the heterologous plant TRXs responsible of this complementation is a type-II PRX.⁵ We think that PsTRXh2 could interact with this yeast PRX blocking it and producing the hypersensitivity. In fact, we have found a similar effect in other protein targeted in vitro by TRXs. In the (Fig. 1), we present the in vitro ability of PsTRXh1 and PsTRXh2 to reduce and activate the pea chloroplastic fructose-1,6-bisphosphatase (FBPase). In the presence of PsTRXh1, FBPase presents TRX-dependent activity but lower than that found when chloroplastic f and m1 isoforms are used.¹² On the opposite, PsTRXh2 presents no only FBPase activation capability but its presence induces the FBPase inhibition, as the enzymatic activity was lower than that exhibited by this enzyme without TRX.Open in a separate windowFigure 1TRX-dependent chloroplastic FBPase activity two-step procedure.¹³ , PsTRXf; ▴, PsTRXm1; •, PsTRXh1; , PsTRXh2. The negative control (no TRX) is represented by a symbol-less line. Numbers in each line represent maximum enzymatic velocity as OD₃₄₀/min.Considering all data, we think that the behaviour showed by both pea h-type TRXs is due to their interactions with several protein-targets, as the PRXs: when the ROS levels increase drastically, cells develop high redox imbalances or even undergo oxidative stress. In this situation, all antioxidant mechanisms must be activated, including the increase of PsTRXh1 expression and protein quantities, giving rise to a more efficient cell detoxification. Under nonimbalance conditions of the redox status, PsTRXh2 could act by interacting (activating or inhibiting) with some protein targets. However, the physiological target for PsTRXh2 are not yet described nor supposed. Our results suggest that its role in the redox control is by producing sensitivity to oxidant agents, maybe by allowing physiological ROS levels in cells.     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Complete Genome Sequence of Croceibacter atlanticus HTCC2559T

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559^T, which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559^T contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559^T, which identified it as an obligate chemoheterotroph.The phylum Bacteroidetes comprises 6 to ∼30% of total bacterial communities in the ocean by fluorescence in situ hybridization (8-10). Most marine Bacteroidetes are in the family Flavobacteriaceae, most of which are aerobic respiratory heterotrophs that form a well-defined clade by 16S rRNA phylogenetic analyses (4). The members of this family are well known for degrading macromolecules, including chitin, DNA, cellulose, starch, and pectin (17), suggesting their environmental roles as detritus decomposers in the ocean (6). Marine Polaribacter and Dokdonia species in the Flavobacteriaceae have also shown to have photoheterotrophic metabolism mediated by proteorhodopsins (11, 12).Several strains of the family Flavobacteriaceae were isolated from the Sargasso Sea and Oregon coast, using high-throughput culturing approaches (7). Croceibacter atlanticus HTCC2559^T was cultivated from seawater collected at a depth of 250 m from the Sargasso Sea and was identified as a new genus in the family Flavobacteriaceae based on its 16S rRNA gene sequence similarities (6). Strain HTCC2559^T met the minimal standards for genera of the family Flavobacteriaceae (3) on the basis of phenotypic characteristics (6).Here we report the complete genome sequence of Croceibacter atlanticus HTCC2559^T. The genome sequencing was initiated by the J. Craig Venter Institute as a part of the Moore Foundation Microbial Genome Sequencing Project and completed in the current announcement. Gaps among contigs were closed by Genotech Co., Ltd. (Daejeon, Korea), using direct sequencing of combinatorial PCR products (16). The HTCC2559^T genome was analyzed with a genome annotation system based on GenDB (14) at Oregon State University and with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (15, 16).The HTCC2559^T genome is 2,952,962 bp long, with 33.9 mol% G+C content, and there was no evidence of plasmids. The number of protein-coding genes was 2,715; there were two copies of the 16S-23S-5S rRNA operon and 36 tRNA genes. The HTCC2559^T genome contained genes for a complete tricarboxylic acid cycle, glycolysis, and a pentose phosphate pathway. The genome also contained sets of genes for metabolic enzymes involved in carotenoid biosynthesis and also a serine/glycine hydroxymethyltransferase, which is often associated with the assimilatory serine cycle (13). The potential for HTCC2559^T to use bacterial type III polyketide synthase (PKS) needs to be confirmed because this organism had a naringenin-chalcone synthase (CHS) or chalcone synthase (EC 2.3.1.74), a key enzyme in flavonoid biosynthesis. CHS initiates the addition of three molecules of malonyl coenzyme A (malonyl-CoA) to a starter CoA ester (e.g., 4-coumaroyl-CoA) (1) and takes part in a few bacterial type III polyketide synthase systems (1, 2, 5, 18).The complete genome sequence confirmed that strain HTCC2559^T is an obligate chemoheterotroph because no genes for phototrophy were found. As expected from physiological characteristics (6), the HTCC2559^T genome contained a set of genes coding for enzymes required to degrade high-molecular-weight compounds, including peptidases, metallo-/serine proteases, pectinase, alginate lyases, and α-amylase.     

Redox regulation of auxin signaling and plant development in Arabidopsis

Thioredoxin (NTR/TRX) and glutathione (GSH/GRX) are the two major systems that play a key role in the maintenance of cellular redox homeostasis. They are essential for plant development, cell division or the response to environmental stresses. In a recent article,¹ we studied the interplay between the NADP-linked thioredoxin and glutathione systems in auxin signaling genetically, by associating TRX reductase (ntra ntrb) and glutathione biosynthesis (cad2) mutations. We show that these two thiol reduction pathways interfere with developmental processes. This occurs through modulation of auxin activity as shown by genetic analyses of loss of function mutations in a triple ntra ntrb cad2 mutant. The triple mutant develops almost normally at the rosette stage but fails to generate lateral organs from the inflorescence meristem, producing almost naked stems that are reminiscent of mutants affected in PAT (polar auxin transport) or biosynthesis. The triple mutant exhibits other defects in processes regulated by auxin, including a loss of apical dominance, vasculature defects and reduced secondary root production. Furthermore, it has lower auxin (IAA) levels and decreased capacity for PAT, suggesting that the NTR and glutathione pathways influence inflorescence meristem development through regulation of auxin transport and metabolism.Key words: arabidopsis, NTS pathway, NGS pathway, thioredoxin (TRX), glutaredoxine (GRX), polar auxin transport (PAT), auxin biosynthesis, pin-like phenotype, apical dominance, meristematic activityExposure of living organisms to environmental stresses triggers various defense and developmental responses. Redox signaling is involved in many aspects of these responses.^2–6 The key players in these responses are the NADPH-dependent glutathione/glutaredoxin system (NGS) and the NADPH-dependent thioredoxin system (NTS). TRX and GRX play key roles in the maintenance of cellular redox homeostasis.^7–10 Genetic approaches aiming to identify functions of TRX and GRX in knock-out plants have largely been limited by the absence of phenotypes of single mutants, presumably due to functional redundancies among members of the multigene families of TRX and GRX.¹¹ Interplay between NTS and NGS pathways have been studied in different organisms^12–17 and association of mutants involved in these two pathways have recently revealed new functions in several aspects of plant development.^4–6     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Arabidopsis glutathione reductase 1 is dually targeted to peroxisomes and the cytosol

We recently established a proteome methodology for Arabidopsis leaf peroxisomes and identified more than 90 putative novel proteins of the organelle. These proteins included glutathione reductase isoform 1 (GR1), a major enzyme of the antioxidative defense system that was previously reported to be cytosolic. In this follow-up study, we validated the proteome data by analyzing the in vivo subcellular targeting of GR1 and the function of its C-terminal tripeptide, TNL>, as a putative novel peroxisome targeting signal type 1 (PTS1). The full-length protein was targeted to peroxisomes in onion epidermal cells when fused N-terminally with the reporter protein. The efficiency of peroxisome targeting, however, was weak upon expression from a strong promoter, consistent with the idea that the enzyme is dually targeted to peroxisomes and the cytosol in vivo. The reporter protein that was extended C-terminally by 10 amino acid residues of GR1 was directed to peroxisomes, characterizing TNL> as a novel PTS1. The data thus identify plant peroxisomal GR at the molecular level in the first plant species and complete the plant peroxisomal ascorbate-glutathione cycle. Moreover, GR1 is the first plant protein that is dually targeted to peroxisomes and the cytosol. The evolutionary origin and regulatory mechanisms of dual targeting are discussed.Key words: ascorbate-glutathione cycle, dual targeting, proteome analyses, reactive oxygen species, targeting signalsMassive amounts of hydrogen peroxide (H₂O₂) are produced during photosynthesis in peroxisomes by glycolate oxidase activity as part of the photorespiratory cycle.¹ Next to catalase, the ascorbate-glutathione cycle is the secondary scavenging system for H₂O₂ detoxification.^2–4 The cycle comprises four enzymes, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and NADPH-dependent glutathione reductase (GR). GR plays a major physiological role in maintaining and regenerating reduced glutathione in response to biotic and abiotic stresses in plants.⁵ Jiminez et al. (1997) provided biochemical evidence for the presence of the antioxidants ascorbate and glutathione and the enzymes of the ascorbate-glutathione cycle in pea peroxisomes.^6–8 While Arabidopsis APX3, MDAR1 and MDAR4 have been characterized as peroxisomal isoforms,^9–11 the molecular identity of plant peroxisomal GR and DHAR have not been determined in any plant species to date.⁵ Arabidopsis encodes two GR and five DHAR isoforms that are either shown to be or predicted to be cytosolic, mitochondrial or plastidic.¹² We recently identified specific isoforms of GR (GR1, At3g24170) and DHAR (DHAR1, At1g19570) as being peroxisome-associated by proteome analysis of Arabidopsis leaf peroxisomes.^13,14 Both isoforms were previously reported to be or predicted to be cytosolic.¹⁵Arabidopsis GR1 terminates with TNL>, which is related to functional plant PTS1 tripeptides such as SNL> and ANL>.^16,17 Threonine (T), however, has not yet been described as an allowed residue at position −3 of PTS1s in any plant peroxisomal protein.¹⁶ Analysis of homologous plant proteins and expressed sequence tags (ESTs) shows that TNL> is generally highly conserved in putative plant GR1 orthologs (Fig. 1). A few other sequences terminate with related tripeptides, such TSL>, TTL>, NNL> and TKL>. Only a single EST (Picrorhiza kurrooa) carries the canonical PTS1, SKI> (Fig. 1). The data provide only weak additional support for peroxisome targeting of plant GR1 orthologs. However, GR homologs from green algae (chlorophyta) carry canonical PTS1 tripeptides, such as SKL> (Chlamydomonas, Volvox) and AKM> (Micromonas, Fig. 1, Suppl. Fig. 1).Open in a separate windowFigure 1Analysis of PTS1 conservation in plant GR1 homologs. Sequences of full-length protein (FLP) plant GR1 homologs or ESTs (“EST”) were identified by BLAST and phylogenetic analysis, aligned by ClustalX, and conserved residues were shaded by Genedoc. In addition to spermatophyta, homologs from bryophyta and chlorophyta were analyzed for PTS1 conservation. For a phylogenetic analysis of the full-length proteins, see also Supplementary Figure 1. The species abbreviations are as follows: Aa, Artemisia annua; At, Arabidopsis thaliana; Bn, Brassica napus; Br, Brassica rapa; Ci, Cichorium intybus; Cr, Chlamydomonas reinhardtii; Cs, Cynara scolymus; Fv, Fragaria vesca; Ha, Helianthus annuus; Msp, Micromonas sp. RCC 299; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pk, Picrorhiza kurrooa; Ppat, Physcomitrella patens subsp. patens; Ps, Pisum sativum; Ptri, Populus trichocarpa; Rc, Ricinus communis; Rs, Raphanus sativus; Tp, Trifolium pratense; Tpus, Triphysaria pusilla; Vc, Volvox carteri f. nagariensis; Vv, Vitis vinifera; Zm, Zea mays.     

Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?

Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs.Key words: membrane occupation and recognition nexus (MORN) domain, phosphatidylinositol phosphate kinase, phosphatidic acid, Physcomitrella patensPhosphoinositides (PIs) are minor membrane phospholipds that play pivotal roles in various signal transduction cascades involved in development and stress response via the regulation of cytoskeletal organization, ion channel activation and vesicle trafficking.^1,2 These are derivatives of phosphatidylinositol (PtdIns) produced by phosphorylation of the 3-, 4- and 5- positions of the inositol ring.² To address the roles of PIs, enzymes involved in their production have been extensively studied using biochemical and molecular biological approaches. Of these enzymes, phosphatidylinositol monophosphate kinases (PIPKs) catalyze the reaction producing phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] that is a substrate of phospholipase C and phosphatidylinositol 3-kinase, and also acts as an intracellular messenger involved in the regulation of F-actin organization and activity of ion channels.^1–3 Although PtdIns(4,5)P₂ is produced by sequential phosphorylation by phosphatidylinositol 4-kinase, producing phosphatidylinositol-4-phosphate [PtdIns(4)P], and then by PIPK,^1,2 the cellular levels of PtdIns(4)P are much higher compared to PtdIns(4,5)P₂.^4–6 Thus, a restriction step controlling cellular PtdIns(4,5)P₂ contents is mediated by PIPKs, indicating the importance of PIPK regulation in various kinds of physiological processes.The roles of plant PIPKs have been established in growth regulation, such as polarized tip growth of root hairs and pollen tubes, via their localization at plasma membranes.^7–12 It is worth to note that plant PIPKs contain a unique structure consisting of a repeat of a membrane occupation recognition nexus (MORN) motifs, called MORN domain, at the N-terminal region and a C-terminal PIPK catalytic domain, except for AtPIP5K10 and AtPIP5K11 from Arabidopsis thaliana, which lack the N-terminal MORN domain.¹³ The MORN domain was first identified as plasma membrane-binding module in junctophilin¹⁴ and the involvement of the MORN domain in plasma membrane localization was proposed for A. thaliana AtPIP5K1 and AtPIP5K3.^9,15,16Another remarkable feature of eukaryotic PIPKs is dependency of the enzymatic activity on phosphatidic acid (PA).^17,18 Indeed, PA-dependent activation of PIPKs has been observed in A. thaliana and in the moss Physcomitrella patens,^6,19,20 as with animal type I PIPKs.²¹ Although much less is known about how PA activates PIPKs in plants, biochemical analyses suggested the involvement of the MORN domain in PA-dependent activation of AtPIP5K1.¹⁵Based on above findings, it was proposed that plasma membrane-localization and PA-dependent activation of plant PIPKs might be regulated by the MORN domain.^9,15,16 In contrast, we recently demonstrated the critical involvement of the C-terminal half containing the catalytic domain of plant PIPKs in both plasma membrane-localization and PA-dependent activation.²² Thus, the function of the MORN domain remains elusive in plant PIPKs.As shown earlier, the N-terminal half of P. patens PpPIPK1 containing the MORN domain enhances its catalytic activity.²² Thus, to identify the region required for the activation of PpPIPK1, we further dissected the N-terminal half into 3 regions; the N-terminal region (amino acid nos. 1–154), the MORN repeat (amino acid nos. 155–316) and the linker region (amino acid nos. 338–452), and made deletion mutants of PpPIPK1 as shown in Figure 1A. Using Pfu Turbo DNA polymerase (Stratagene, La Jolla, USA), DNA fragments corresponding to deletion mutants lacking the N-terminal and N-terminal plus the MORN repeat, designated PpPIPK1ΔN and PpPIPK1ΔN-MORN, respectively, were amplified with primer sets; one is M_PIPK1_fb (5′-GGC AAG CAC GTG TAT AAT GTC TGA AGG GCT T-3′) and XhoIPIPK1 (5′-TAA ACT CGA GTT AGC TGG GTA GGA GGA AA-3′) and the other is M_PIPK1_f7 (5′-AGA GAA CAC GTG TAT AAT GTC TGA CTT CTA CGT CGG T-3′) and XhoIPIPK1. For building an expression plasmid for a deletion mutant lacking the MORN repeat, designated PpPIPK1ΔMORN, the N-terminal region and PpPIPK1ΔN-MORN were amplified with primer sets, M_PIPK1_fb and M_PIPK1_r3 (5′-TTG TAA GTC TCG GGT GCC ATT TGA GAG CTC-3′) M_PIPK1_f6 (5′-GAG CTC TCA AAT GGC ACC CGA GAC TTA CAA-3′) and XhoIPIPK1, respectively, using Pfu Turbo DNA polymerase and resultant DNA fragments were fused by PCR with a primer set, M_PIPK1_fb and XhoIPIPK1 using the same enzyme. These PCR products were digested with Pml1 and XhoI and inserted into Pml1-XhoI digested pPICZB (Invitrogen) to construct expression plasmids, pPICZB-PpPIPK1ΔN, pPICZB-PpPIPK1ΔN-MORN and pPICZB-PpPIPK1ΔMORN. Transformation of P. pastoris X-33 cells with the above expression plasmids, colony PCR of transformants and following expression, purification and western blot analysis of His-tagged recombinant proteins were performed as described previously.⁶ The PIPK activity assay using purified His-tagged proteins was carried out as described previously²³ with the modifications.⁶Open in a separate windowFigure 1Functional dissection of the N-terminal region of PpPIPK1 identifies positive regulatory regions. (A) His-tagged recombinant PpPIPK1 proteins. A repetition of eight MORN motifs (grey boxes) and the conserved catalytic domain (black box) are indicated in wild type and mutant PpPIPK1s. The MORN repeat and junction of internal deletion are indicated by amino acid position numbers. (B) In vitro lipid kinase activity of His-tagged recombinant proteins. The activities of recombinant proteins bound to Ni-NTA agarose beads were assayed with PtdIns4P. (C) In vitro PA-dependent lipid kinase activity of His-tagged proteins. The activities of recombinant proteins bound to Ni-NTA agarose beads were assayed with PtdIns4P with 143 µM PA. Top and bottom arrowheads represent reaction products PtdIns(4,5)P₂ and lysoPtdIns(4,5)P₂, respectively.Biochemical analyses of these enzymes after expression in yeast P. pastoris X-33 cells followed by purification showed that deletion of the N-terminal region (PpPIPK1ΔN) reduced PpPIPK1 activity ca 40% compared to the full length enzyme, whereas loss of the MORN repeat (PpPIPK1ΔMORN) had no significant effect (Fig. 1B). In agreement, a mutant lacking four MORN repeats of the total eight repeats showed no difference in the activity compared the full length enzyme (data not shown). These results indicate a positive role of the N-terminal region, but not the MORN repeats, on PpPIPK1 activity. However, these findings differ from those obtained with AtPIP5K1, where the MORN domain represses enzymatic activity.¹⁵ Interestingly, PpPIPK1ΔN-MORN containing the linker and catalytic regions showed higher enzymatic activity of ca 23 % compared to the full length PpPIPK1 (Fig. 1B). The C-terminal half only containing the catalytic domain of PpPIPK1 and thus lacking the linker region showed a reduced activity.²² It is therefore proposed that the linker region carries a positive regulatory element. Although details are unknown, negligible effects of the N-terminal and MORN domains for the enzymatic activity has been indicated in AtPIP5K3 from A. thaliana.¹¹ Moreover, it is noteworthy that PA-dependent activation was not affected by any deletion as shown in Figure 1C, confirming that the N-terminal half is not involved in PA dependency of the PpPIPK1 activity.²²Our results indicated that the MORN domain is not involved in the regulation of the catalytic activity in PpPIPK1. Similarly, the function of the MORN domain found in the accumulation and replication of chloroplasts 3 (ARC3) was not resolved. ARC3 is an FtsZ homologue involved in chloroplast division²⁴ and the only protein containing the MORN repeats other than PIPKs in A. thaliana. It was shown that the ARC3 MORN domain did not interact with any stromal plastid division components.²⁵ Moreover, there are reports representing functions of the MORN domain other than plasma membrane binding. Human amyotrophic lateral sclerosis 2 (ALS2), a guanine nucleotide exchange factor (GEF) specific to the small GTPase Rab5, contains the MORN domain at the central region that is essential for the GEF activity but not for interaction with Rab5.²⁶ In contrast, specific interaction of the MORN domain with Rab-E GTPases and resultant enzymatic activation was recently demonstrated for AtPIP5K2.¹² It is interesting that these results are inconsistent with each other in terms of interaction of the MORN domain with small GTPases.Taken together, with no function of the MORN domain in plasma membrane localization of PpPIPK1 and AtPIP5K1,²² the function of the MORN domain is still unknown, despite its high conservation plants PIPKs. Alternatively, based on the findings of ARC3, ALS2 and AtPIP5K2,^12,25,26 the function of the MORN domain possibly varies among PIPK isoforms and may thus have multifunctional roles. Therefore, it is necessary to identify interaction partners for the MORN domain of each plant PIPKs and to analyze phenotypes of transgenic plants carrying MORN domain-lacking PIPKs during developmental process and environmental stress responses.     

Myosin light chain kinases: Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.¹ A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.²One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.^1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.² Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.⁴ Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.⁵ Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.⁶ More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.^7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.^8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.^10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).¹¹ Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,¹¹ have been shown to colocalize on stress fibers.^12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.^14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.¹⁶ More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.¹⁷ The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.^9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.^17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.^17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.^20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.¹⁷ These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,^11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.¹¹Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).^23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.^8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.^23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.^26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,^26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),^18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,²⁹ MLCK only causes diphosphorylation when present at higher concentrations.³⁰ By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.^30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.^21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear²⁵ and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.²³ Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.¹⁷ These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.^11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.¹⁷The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.³⁴ GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,³⁵ and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.     

Does OsPHR2, central Pi-signaling regulator,regulate some unknown factors crucial for plant growth?

OsPHR2, the homolog of AtPHR1, is a central Pi-signaling regulator. The Pi-signaling pathway downstream of AtPHR1, similarly of OsPHR2,^1,2 involves a noncoding RNA which targets mimicry of miR399. miRNA399 mediates cleavage of PHO2.^3,4 The regulating pathway downstream of OsPHR2 is negatively regulated by the Pi-signaling responsive gene OsSPX1.^5,6 Overexpression of AtPHR1 and OsPHR2 leads to an increased concentration of Pi in the shoot tissues with leaf toxic symptom and growth retardation similar as the phenotype of pho2 mutant, especially under Pi abundant conditions.^2,6,7 It has been known that the low affinity Pi transporter OsPT2 mainly contributes to the shoot Pi accumulation mediated by OsPHR2, and overexpression of OsPT2 results in shoot Pi accumulation and leaf toxic symptom and growth retardation under Pi abundant conditions.⁶ Two curious questions are emerging from the reported results: How Os SPX1 functions on the negative regulation of the pathway and what mechanism of the growth retardation mediated by OsPHR2. For the second question, our favored hypothesis is that the growth inhibition mediated by overexpression of OsPHR2 is caused by toxic physiological effects due to excessive Pi accumulation in shoots (Pi toxicity). In fact, the toxic symptoms become diminished with decreased Pi levels in growth medium. However, the plant growth retardation mediated by overexpression of OsPHR2 may be caused by some unknown genetic factor(s) regulated by OsPHR2.Key words: Oryza Sativa L, OsPHR2, OsSPX1, pi-signaling, plant growth