Chlorophyll f production in two new subaerial cyanobacteria of the family Oculatellaceae.

Chlorophyll (Chl) f was recently identified in a few cyanobacteria as the fifth chlorophyll of oxygenic organisms. In this study, two Leptolyngbya-like strains of CCNU0012 and CCNU0013 were isolated from a dry ditch in Chongqing city and a brick wall in Mount Emei Scenic Area in China, respectively. These two strains were described as new species: Elainella chongqingensis sp. nov. (Oculatellaceae, Synechococcales) and Pegethrix sichuanica sp. nov. (Oculatellaceae, Synechococcales) by the polyphasic approach based on morphological features, phylogenetic analysis of 16S rRNA gene and secondary structure comparison of 16S-23S internal transcribed spacer domains. Both strains produced Chl a under white light (WL) but additionally induced Chl f synthesis under far-red light (FRL). Unexpectedly, the content of Chl f in P. sichuanica was nearly half that in most Chl f-producing cyanobacteria. Red-shifted phycobiliproteins were also induced in both strains under FRL conditions. Subsequently, additional absorption peak beyond 700 nm in the FRL spectral region appeared in these two strains. This is the first report of Chl f production induced by FRL in the family Oculatellaceae. This study not only extended the diversity of Chl f-producing cyanobacteria but also provided precious samples to elucidate the essential binding sites of Chl f within cyanobacterial photosystems.

Kovacikia minuta sp. nov. (Leptolyngbyaceae, Cyanobacteria), a new freshwater chlorophyll f-producing cyanobacterium.

A few groups of cyanobacteria have been characterized as having far-red light photoacclimation (FaRLiP) that results from chlorophyll f (Chl f) production. In this study, using a polyphasic approach, we taxonomically transferred the Cf. Leptolyngbya sp. CCNUW1 isolated from a shaded freshwater pond, which produces Chl f under far-red light, to the genus Kovacikia and named this taxon Kovacikia minuta sp. nov. This strain was morphologically similar to Leptolyngbya-like strains. The thin filaments were purplish-brown under white light but became grass green under far-red light. The 31-gene phylogeny grouped K. minuta CCNU0001 into order Synechococcales and family Leptolyngbyaceae. Phylogenetic analysis based on 16S rRNA gene sequences further showed that K. minuta CCNU0001 was clustered into Kovacikia with similarities of 97.2-97.4% to the recently reported type species of Kovacikia muscicola HA7619-LM3. Additionally, the internal transcribed spacer region between 16S-23S rRNA genes had a unique sequence and secondary structure compared with other Kovacikia strains and phylogenetically related taxa. Draft genome sequences of K. minuta CCNU0001 (8,564,336 bp) were assembled into one circular chromosome and two circular plasmids. A FaRLiP 20-gene cluster comprised two operons with the unique organization. In sum, K. minuta was established as a new species, and it is the first species reported to produce Chl f and for which a draft genome was produced in genus Kovacikia. This study expanded our knowledge regarding the diversity of Chl f-producing cyanobacteria in far-red light-enriched environments and provides important foundational information for future investigations of FaRLiP evolution in cyanobacteria.

New types of ATP-grasp ligase are associated with the novel pathway for complicated mycosporine-like amino acid production in desiccation-tolerant cyanobacteria.

Mycosporine-like amino acids (MAAs) were widespread in diverse organisms to attenuate UV radiation. We recently characterized the large, complicated MAA mycosporine-2-(4-deoxygadusolyl-ornithine) in desert cyanobacterium Nostoc flagelliforme. Synthesis of this MAA requires the five-gene cluster mysABDC2C3. Here, bioinformatic analysis indicated that mysC duplication within five-gene mys clusters is strictly limited to drought-tolerant cyanobacteria. Phylogenic analysis distinguished these duplicated MysCs into two clades that separated from canonical MysCs. Heterologous expression of N. flagelliforme mys genes in Escherichia coli showed that MysAB produces 4-deoxygadusol. The ATP-grasp ligase of MysC3 catalyses the linkage of the δ- or ε-amino group of ornithine/lysine to 4-deoxygadusol, yielding mycosporine-ornithine or mycosporine-lysine respectively. The ATP-grasp ligase of MysC2 strictly condenses the α-amino group of mycosporine-ornithine to another 4-deoxygadusol. MysD (D-Ala-D-Ala ligase) functions following MysC2 to catalyse the formation of mycosporine-2-(4-deoxygadusolyl-ornithine). High arginine content likely provides a greater pool of ornithine over other amino acids during rehydration of desiccated N. flagelliforme. Duplication of ATP-grasp ligases is specific for the use of substrates that have two amino groups (such as ornithine) for the production of complicated MAAs with multiple chromophores. This five-enzyme biosynthesis pathway for complicated MAAs is a novel adaptation of cyanobacteria for UV tolerance in drought environments.

Identification and characterization of SaeIF1 from the eukaryotic translation factor SUI1 family in cadmium hyperaccumulator Sedum alfredii.

MAIN CONCLUSION: Cadmium-sensitive yeast screening resulted in the isolation of protein translation factor SaeIF1 from the hyperaccumulator Sedum alfredii which has both general and special regulatory roles in controlling cadmium accumulation. The hyperaccumulator of Sedum alfredii has the extraordinary ability to hyperaccumulate cadmium (Cd) in shoots. To investigate its underlying molecular mechanisms of Cd hyperaccumulation, a cDNA library was generated from leaf tissues of S. alfredii. SaeIF1, belonging to the eukaryotic protein translation factor SUI1 family, was identified by screening Cd-sensitive yeast transformants with this library. The full-length cDNA of SaeIF1 has 582 bp and encodes a predicted protein with 120 amino acids. Transient expression assays showed subcellular localization of SaeIF1 in the cytoplasm. SaeIF1 was constitutively and highly expressed in roots and shoots of the hyperaccumulator of S. alfredii, while its transcript levels showed over 100-fold higher expression in the hyperaccumulator of S. alfredii relative to the tissues of a nonhyperaccumulating ecotype of S. alfredii. However, the overexpression of SaeIF1 in yeast cells increased Cd accumulation, but conferred more Cd sensitivity. Transgenic Arabidopsis thaliana expressing SaeIF1 accumulated more Cd in roots and shoots without changes in the ratio of Cd content in shoots and roots, but were more sensitive to Cd stress than wild type. Both special and general roles of SaeIF1 in Cd uptake, transportation, and detoxification are discussed, and might be responsible for the hyperaccumulation characteristics of S. alfredii.

Widespread occurrence and unexpected diversity of red-shifted chlorophyll producing cyanobacteria in humid subtropical forest ecosystems.

Discovery of red-shifted chlorophyll d and f in cyanobacteria has opened up new avenues to estimate global carbon fixation driven by far-red light. Shaded habitats in humid subtropical forest ecosystems contain an increased proportion of far-red light components relative to residual white light. After an extensive survey of shaded ecosystems within subtropical forests, wide occurrence of red-shifted chlorophyll-producing cyanobacteria was demonstrated by isolated Chl f-producing and Chl d-containing cyanobacteria. Chl f-producing cyanobacteria were classified into the genera of Aphanocapsa and Chroococcidiopsis and two undescribed genera within Leptolyngbyaceae. Newly isolated Chl d-containing Acaryochloris sp. CCNUM4 showed the closest phylogenetic relationship with Acaryochloris species isolated from marine environments. Acaryochloris sp. CCNUM4 produced Chl d as major photopigment, and Chl f-producing cyanobacteria use Chl a under white light conditions but Chl a + f under far-red light conditions. Their habitats are widely distributed in subtropical forest ecosystems and varied from mosses on limestone to macrophyte and freshwater in the streams and ponds. This study presents a significant advance in the knowledge of distribution and diversity of red-shifted chlorophyll-producing cyanobacteria in terrestrial ecosystems. The results suggest that Chl f-producing and Chl d-containing cyanobacteria might be important primary producers in far-red light dominant niches worldwide.

Genomic and transcriptomic insights into the survival of the subaerial cyanobacterium Nostoc flagelliforme in arid and exposed habitats.

The cyanobacterium Nostoc flagelliforme is an extremophile that thrives under extraordinary desiccation and ultraviolet (UV) radiation conditions. To investigate its survival strategies, we performed whole-genome sequencing of N. flagelliforme CCNUN1 and transcriptional profiling of its field populations upon rehydration in BG11 medium. The genome of N. flagelliforme is 10.23 Mb in size and contains 10 825 predicted protein-encoding genes, making it one of the largest complete genomes of cyanobacteria reported to date. Comparative genomics analysis among 20 cyanobacterial strains revealed that genes related to DNA replication, recombination and repair had disproportionately high contributions to the genome expansion. The ability of N. flagelliforme to thrive under extreme abiotic stresses is supported by the acquisition of genes involved in the protection of photosynthetic apparatus, the formation of monounsaturated fatty acids, responses to UV radiation, and a peculiar role of ornithine metabolism. Transcriptome analysis revealed a distinct acclimation strategy to rehydration, including the strong constitutive expression of genes encoding photosystem I assembly factors and the involvement of post-transcriptional control mechanisms of photosynthetic resuscitation. Our results provide insights into the adaptive mechanisms of subaerial cyanobacteria in their harsh habitats and have important implications to understand the evolutionary transition of cyanobacteria from aquatic environments to terrestrial ecosystems.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

The identification of IsiA proteins binding chlorophyll d in the cyanobacterium Acaryochloris marina.

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (-F6) isolated from iron-deficient culture contained Chl d-bound PSI-IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).

UV-B induced biosynthesis of a novel sunscreen compound in solar radiation and desiccation tolerant cyanobacteria.

The small-molecule sunscreen compounds, mycosporine-like amino acids (MAAs), have strong ultraviolet (UV) absorption and can protect cyanobacteria against UV-B damage. However, the molecular mechanism underlying UV-B signaling and MAA chemical diversity remain largely unclear. Here, we identified a five-gene cluster for MAA biosynthesis in the solar radiation and desiccation tolerant cyanobacterium Nostoc flagelliforme. A LuxR family protein OrrA was identified as a positive UV-B responsive regulator binding to the promoter region of this gene cluster. OrrA functions as an activator mediating the UV-B induced MAA biosynthesis. Overexpression of orrA strengthened its UV-B tolerance during desiccation, and enhanced the photosynthetic recovery upon rehydration. Heterologous expression of this gene cluster in Anabaena PCC 7120 produces the same MAA as that in field samples of N. flagelliforme. The MAA structure is assigned as mycosporine-2-(4-deoxygadusolyl-ornithine) with a molecular weight of 756 Da, the structurally unique MAA compound reported to date. This MAA was catalyzed by mysD-mysC2-mysC1 encoding proteins from 4-deoxygadusol, which was synthesized through the catalysis of mysA-mysB products. Thus, we elucidated the transcriptional mechanism for a novel type MAA biosynthesis in solar radiation and desiccation tolerant cyanobacteria, which shed light on the identification of other components for UV-B signaling in cyanobacteria.

Quantitative proteomics analysis of leaves from two Sedum alfredii (Crassulaceae) populations that differ in cadmium accumulation.

Due to their extraordinary capacity to hypertolerate and hyperaccumulate heavy metals in above-ground tissues, hyperaccumulator plant species have gained wide attention from researchers seeking biotechnologies to manage environmental heavy metal pollution. However, the molecular basis of hyperaccumulation is still far from being fully understood. Here, we used iTRAQ to perform a quantitative proteomics study of the leaves of Sedum alfredii (Crassulaceae) from hyperaccumulating population (HP) and non-hyperaccumulating population (NHP). A total of 248 proteins had constitutively higher levels in HP leaves than in NHP leaves. Cadmium (Cd) treatment led to the induction of 13 proteins in HP leaves and 33 proteins in NHP leaves. Two proteins were induced by Cd in both HP leaves and NHP leaves. The annotations for many of the proteins that were higher in HP leaves and proteins that were induced by Cd treatments were associated with vacuolar sequestration, cell wall/membrane modification, and plant defense. In addition to establishing a global empirical foundation for the study of proteins in S. alfredii, our findings relating to the differential constitutive and inducible expression of proteins open potential new research avenues and bolster previously reported suppositions about Cd hyperaccumulation in hyperaccumulator plants.

Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

BACKGROUND: Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). RESULTS: In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed miRNAs (DEMs) and DEGs revealed 92 mRNA-miRNA interactions between CL and DL plants, and 32 mRNA-miRNA interactions between DL and WL plants. CONCLUSIONS: This study provides a global view of the transcriptional and the post-transcriptional responses of tobacco under drought stress and re-watering conditions. Our results establish an empirical foundation that should prove valuable for further investigations into the molecular mechanisms through which tobacco, and plants more generally, respond to drought stress at multiple molecular genetic levels.

Enhanced cadmium efflux and root-to-shoot translocation are conserved in the hyperaccumulator Sedum alfredii (Crassulaceae family).

Investigation on the molecular mechanisms of cadmium hyperaccumulation has been mostly focused on members of the Brassicaceae family. Here, we show using hyperaccumulating (HP) and nonhyperaccumulating (NHP) populations of Sedum alfredii (Crassulaceae), that Cd hypertolerance correlates with higher Cd efflux rates and less cadmium accumulation in suspension cells and roots. The heavy metal ATPase HMA2, but not HMA4, was highly expressed in suspension cultures and roots from HP plants compared to NHP cells and plants. Reciprocal grafting also showed that Cd translocation is more efficient in HP plants. These results suggest that cadmium efflux is a conserved mechanism among natural cadmium hyperaccumulator species.

Inter-laboratory validation of the in-vivo flow cytometric micronucleus analysis method (MicroFlow®) in China.

Although inter-laboratory validation efforts of the in-vivo micronucleus (MN) assay based on flow cytometry (FCM) have taken place in the EU and US, none have been organized in China. Therefore, an inter-laboratory study that included eight laboratories in China and one experienced reference laboratory in the US was coordinated to validate the in-vivo FCM MicroFlow(®) method to determine the frequency of micro-nucleated reticulocytes (MN-RETs) in rat blood. Assay reliability and reproducibility were evaluated with four known genotoxicants, and the results obtained with the FCM method were compared with the outcome of the traditional evaluation of bone-marrow micronuclei by use of microscopy. Each of the four chemicals was tested at three sites (two in China and the one US reference laboratory). After three consecutive daily exposures to a genotoxicant, blood and bone-marrow samples were obtained from rats 24h after the third dose. MN-RET frequencies were measured in 20,000 RET in blood by FCM, and micro-nucleated polychromatic erythrocyte (MN-PCE) frequencies were measured in 2,000 PCEs in bone marrow by microscopy. For both methods, each genotoxicant was shown to induce a statistically significant increase in the frequency of MN after treatment with at least one dose. Where more doses than one caused an increase, responses occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM-based MN-RET vs microscopy-based MN-PCE measurements (eight experiments, 200 paired measurements) was 0.723, indicating a high degree of correspondence between methods and compartments. The rs value for replicate FCM MN-RET measurements performed at the eight collaborative laboratories was 0.940 (n=200), and between the eight FCM laboratories with the reference laboratory was 0.933 (n=200), suggesting that the automated method is very well transferable between laboratories. The FCM micronucleus analysis method is currently used in many countries worldwide, and these data support its use for evaluating the in-vivo genotoxic potential of test chemicals in China.

Effects of UV-B radiation and periodic desiccation on the morphogenesis of the edible terrestrial cyanobacterium Nostoc flagelliforme.

The terrestrial cyanobacterium Nostoc flagelliforme Berk. et M. A. Curtis has been a popular food and herbal ingredient for hundreds of years. To meet great market demand and protect the local ecosystem, for decades researchers have tried to cultivate N. flagelliforme but have failed to get macroscopic filamentous thalli. In this study, single trichomes with 50 to 200 vegetative cells were induced from free-living cells by low light and used to investigate the morphogenesis of N. flagelliforme under low UV-B radiation and periodic desiccation. Low-fluence-rate UV-B (0.1 W m(-2)) did not inhibit trichome growth; however, it significantly increased the synthesis of extracellular polysaccharides and mycosporine-like amino acids and promoted sheath formation outside the trichomes. Under low UV-B radiation, single trichomes developed into filamentous thalli more than 1 cm long after 28 days of cultivation, most of which grew separately in liquid BG11 medium. With periodic desiccation treatment, the single trichomes formed flat or banded thalli that grew up to 2 cm long after 3 months on solid BG11 medium. When trichomes were cultivated on solid BG11 medium with alternate treatments of low UV-B and periodic desiccation, dark and scraggly filamentous thalli that grew up to about 3 cm in length after 40 days were obtained. In addition, the cultivation of trichomes on nitrogen-deficient solid BG11 medium (BG11(0)) suggested that nitrogen availability could affect the color and lubricity of newly developed thalli. This study provides promising techniques for artificial cultivation of N. flagelliforme in the future.

Spatial regulation of cyclic AMP-Epac1 signaling in cell adhesion by ERM proteins.

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non-resistant plants.

Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non-resistant plants, but roles for PCs in hyper-accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed-phase high-performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time-dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non-resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii.

Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity.

BACKGROUND: Retrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks. RESULTS: Here we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12th and 59th amino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio in vivo, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen. CONCLUSIONS: Together, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitt's lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS) warrants further investigation into its role in disease development.

Detection of phytochelatins in the hyperaccumulator Sedum alfredii exposed to cadmium and lead.

Phytochelatins (PCs) are known to play an essential role in the heavy metal detoxification of some higher plants and fungi by chelating heavy metals. However, three recent papers reported that no PCs could be detected in the hyperaccumulator Sedum alfredii Hance upon cadmium, lead or zinc treatment, respectively. In this paper, PC synthesis was assayed again in the mine population of S. alfredii with the help of reversed phase high-performance liquid chromatography (HPLC), HPLC-mass spectrometry, and HPLC-tandem mass spectrometry. Our data showed that PC formation could be induced in the leaf, stem and root tissues of S. alfredii upon exposure to 400 microM cadmium, and only in the stem and root when exposed to 700 microM lead. However, no PCs were found in any part of S. alfredii when it was subjected to exposure to 1600 microM zinc.

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner.

Arap3 is a phosphoinositide (PI) 3 kinase effector that serves as a GTPase activating protein (GAP) for both Arf and Rho G-proteins. The protein has multiple pleckstrin homology (PH) domains that bind preferentially phosphatidyl-inositol-3,4,5-trisphosphate (PI(3,4,5,)P3) to induce translocation of Arap3 to the plasma membrane upon PI3K activation. Arap3 also contains a Ras association (RA) domain that interacts with the small G-protein Rap1 and a sterile alpha motif (SAM) domain of unknown function. In a yeast two-hybrid screen for new interaction partners of Arap3, we identified the PI 5'-phosphatase SHIP2 as an interaction partner of Arap3. The interaction between Arap3 and SHIP2 was observed with endogenous proteins and shown to be mediated by the SAM domain of Arap3 and SHIP2. In vitro, these two domains show specificity for a heterodimeric interaction. Since it was shown previously that Arap3 has a higher affinity for PI(3,4,5,)P3 than for PI(3,4)P2, we propose that the SAM domain of Arap3 can function to recruit a negative regulator of PI3K signaling into the effector complex.