SMART MODE:

NORMAL GENOMIC

• Sui N, Yang Z, Liu M, Wang B
• Identification and transcriptomic profiling of genes involved in increasing sugar content during salt stress in sweet sorghum leaves.
• BMC Genomics. 2015; 16: 534-534
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• BACKGROUND: Sweet sorghum is an annual C4 crop considered to be one of the most promising bio-energy crops due to its high sugar content in stem, yet it is poorly understood how this plant increases its sugar content in response to salt stress. In response to high NaCl, many of its major processes, such as photosynthesis, protein synthesis, energy and lipid metabolism, are inhibited. Interestingly, sugar content in sweet sorghum stems remains constant or even increases in several salt-tolerant species. RESULTS: In this study, the transcript profiles of two sweet sorghum inbred lines (salt-tolerant M-81E and salt-sensitive Roma) were analyzed in the presence of 0 mM or 150 mM NaCl in order to elucidate the molecular mechanisms that lead to higher sugar content during salt stress. We identified 864 and 930 differentially expressed genes between control plants and those subjected to salt stress in both M-81E and Roma strains. We determined that the majority of these genes are involved in photosynthesis, carbon fixation, and starch and sucrose metabolism. Genes important for maintaining photosystem structure and for regulating electron transport were less affected by salt stress in the M-81E line compared to the salt-sensitive Roma line. In addition, expression of genes encoding NADP+-malate enzyme and sucrose synthetase was up-regulated and expression of genes encoding invertase was down-regulated under salt stress in M-81E. In contrast, the expression of these genes showed the opposite trend in Roma under salt stress. CONCLUSIONS: The results we obtained revealed that the salt-tolerant genotype M-81E leads to increased sugar content under salt stress by protecting important structures of photosystems, by enhancing the accumulation of photosynthetic products, by increasing the production of sucrose synthetase and by inhibiting sucrose decomposition.

• Wang S, Wang J, Yao W, Zhou B, Li R, Jiang T
• Expression patterns of WRKY genes in di-haploid Populus simonii x P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing.
• Plant Cell Rep. 2014; 33: 1687-96
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• KEY MESSAGE: Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii x P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

• Gyulai G et al.
• Phytoextraction potential of wild type and 35S-gshI transgenic poplar trees (Populus x Canescens) for environmental pollutants herbicide paraquat, salt sodium, zinc sulfate and nitric oxide in vitro.
• Int J Phytoremediation. 2014; 16: 379-96
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• Phytoextraction potentials of two transgenic (TR) poplar (Populus x canescens) clones TRggs11 and TRlgl6 were compared with that of wild-type (WT) following exposure to paraquat, zinc sulfate, common salt and nitric oxide (NO), using a leaf-disc system incubated for 21 days on EDTA-containing nutritive WPM media in vitro. Glutathione (GSH) contents of leaf discs of TRlgl6 and TRggs11 showed increments to 296% and 190%, respectively, compared with WT. NO exposure led to a twofold GSH content in TRlgl6, which was coupled with a significantly increased sulfate uptake when exposed to 10(-3) M ZnSO4. The highest mineral contents of Na, Zn, Mn, Cu, and Mo was observed in the TRggs11 clone. Salt-induced activity of catalase enzyme increased in both TR clones significantly compared with WT under NaCl (0.75% and 1.5%) exposure. The in silico sequence analyses of gsh1 genes revealed that P. x canadensis and Salix sachalinensis show the closest sequence similarity to that of P. x canescens, which predicted an active GSH production with high phytoextraction potentials of these species with indication for their use where P. x canescens can not be grown.

• Chen J et al.
• Deep-sequencing transcriptome analysis of low temperature perception in a desert tree, Populus euphratica.
• BMC Genomics. 2014; 15: 326-326
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• BACKGROUND: Compared with other Populus species, Populus euphratica Oliv. exhibits better tolerance to abiotic stress, especially those involving extreme temperatures. However, little is known about gene regulation and signaling pathways involved in low temperature stress responses in this species. Recent development of Illumina/Solexa-based deep-sequencing technologies has accelerated the study of global transcription profiling under specific conditions. To understand the gene network controlling low temperature perception in P. euphratica, we performed transcriptome sequencing using Solexa sequence analysis to generate a leaf transcriptome at a depth of 10 gigabases for each sample. RESULTS: Using the Trinity method, 52,081,238 high-quality trimmed reads were assembled into a non-redundant set and 108,502 unigenes with an average length of 1,047 bp were generated. After performing functional annotations by aligning all-unigenes with public protein databases, 85,584 unigenes were annotated. Differentially expressed genes were investigated using the FPKM method by applying the Benjamini and Hochberg corrections. Overall, 2,858 transcripts were identified as differentially expressed unigenes in at least two samples and 131 were assigned as unigenes expressed differently in all three samples. In 4 degrees C-treated sample and -4 degrees C-treated sample, 1,661 and 866 differently expressed unigenes were detected at an estimated absolute log2-fold change of > 1, respectively. Among them, the respective number of up-regulated unigenes in C4 and F4 sample was 1,113 and 630, while the respective number of down-regulated ungenes is 548 and 236. To increase our understanding of these differentially expressed genes, we performed gene ontology enrichment and metabolic pathway enrichment analyses. A large number of early cold (below or above freezing temperature)-responsive genes were identified, suggesting that a multitude of transcriptional cascades function in cold perception. Analyses of multiple cold-responsive genes, transcription factors, and some key transduction components involved in ABA and calcium signaling revealed their potential function in low temperature responses in P. euphratica. CONCLUSIONS: Our results provide a global transcriptome picture of P. euphratica under low temperature stress. The potential cold stress related transcripts identified in this study provide valuable information for further understanding the molecular mechanisms of low temperature perception in P. euphratica.

• Guo X, Yin H, Cong J, Dai Z, Liang Y, Liu X
• RubisCO gene clusters found in a metagenome microarray from acid mine drainage.
• Appl Environ Microbiol. 2013; 79: 2019-26
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• The enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (cbbL). Forty-six cbbL-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities of cbbL and the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny.

• Li P et al.
• Brassinosteroids-Induced Systemic Stress Tolerance was Associated with Increased Transcripts of Several Defence-Related Genes in the Phloem in
• PLoS One. 2013; 8: 66582-66582
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• Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.

• Ma T et al.
• Genomic insights into salt adaptation in a desert poplar.
• Nat Commun. 2013; 4: 2797-2797
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• Despite the high economic and ecological importance of forests, our knowledge of the genomic evolution of trees under salt stress remains very limited. Here we report the genome sequence of the desert poplar, Populus euphratica, which exhibits high tolerance to salt stress. Its genome is very similar and collinear to that of the closely related mesophytic congener, P. trichocarpa. However, we find that several gene families likely to be involved in tolerance to salt stress contain significantly more gene copies within the P. euphratica lineage. Furthermore, genes showing evidence of positive selection are significantly enriched in functional categories related to salt stress. Some of these genes, and others within the same categories, are significantly upregulated under salt stress relative to their expression in another salt-sensitive poplar. Our results provide an important background for understanding tree adaptation to salt stress and facilitating the genetic improvement of cultivated poplars for saline soils.

• Han Y et al.
• Populus euphratica XTH overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants.
• J Exp Bot. 2013; 64: 4225-38
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• Populus euphratica is a salt-tolerant tree species that develops leaf succulence after a prolonged period of salinity stress. In the present study, a putative xyloglucan endotransglucosylase/hydrolase gene (PeXTH) from P. euphratica was isolated and transferred to tobacco plants. PeXTH localized exclusively to the endoplasmic reticulum and cell wall. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH overexpression. Compared with the wild type, PeXTH-transgenic plants contained 36% higher water content per unit area and 39% higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a 47-78% greater net photosynthesis under control and salt treatments (100-150 mM NaCl). Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling.

• Li B, Duan H, Li J, Deng XW, Yin W, Xia X
• Global identification of miRNAs and targets in Populus euphratica under salt stress.
• Plant Mol Biol. 2013; 81: 525-39
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• Populus euphratica, a typical hydro-halophyte, is ideal for studying salt stress responses in woody plants. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that fulfilled an important post-transcriptional regulatory function. MiRNA may regulate tolerance to salt stress but this has not been widely studied in P. euphratica. In this investigation, the small RNAome, degradome and transcriptome were studied in salt stress treated P. euphratica by deep sequencing. Two hundred and eleven conserved miRNAs between Populus trichocarpa and P. euphratica have been found. In addition, 162 new miRNAs, belonging to 93 families, were identified in P. euphratica. Degradome sequencing experimentally verified 112 targets that belonged to 51 identified miRNAs, few of which were known previously in P. euphratica. Transcriptome profiling showed that expression of 15 miRNA-target pairs displayed reverse changing pattern under salt stress. Together, these results indicate that, in P. euphratica under salt stress, a large number of new miRNAs could be discovered, and both known and new miRNA were functionally cleaving to their target mRNA. Expression of miRNA and target were correspondingly induced by salt stress but that it was a complex process in P. euphratica.

• Chen S, Jiang J, Li H, Liu G
• The salt-responsive transcriptome of Populus simonii x Populus nigra via DGE.
• Gene. 2012; 504: 203-12
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• In this study, the dynamic transcriptome of poplar (Populus simonii x Populus nigra) was investigated under salt stress using Solexa/illumine digital gene expression (DGE) technique. A total of 5453, 2372, and 1770 genes were shown to be differentially expressed after exposure to NaCl for 3 days, 6 days and 9 days, respectively. Differential expression patterns throughout salt stress were identified for 572 genes. Gene ontology classification analysis of these differentially expressed genes revealed that numerous genes mapped to "transporter activity" and "response to stress". The dynamic transcriptome expression profiles of poplar under salt stress obtained in this study may provide useful insights for further analysis of the mechanism of high salinity tolerance in plants. Furthermore, these differentially expressed genes under salt stress may allow identification of potential genes as suitable targets for biotechnological manipulation with the aim of improving poplar salt tolerance.

• Sorokin DY, Muntyan MS, Panteleeva AN, Muyzer G
• Thioalkalivibrio sulfidiphilus sp. nov., a haloalkaliphilic, sulfur-oxidizing gammaproteobacterium from alkaline habitats.
• Int J Syst Evol Microbiol. 2012; 62: 1884-9
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• A moderately salt-tolerant and obligately alkaliphilic, chemolithoautotrophic sulfur-oxidizing bacterium, strain HL-EbGr7(T), was isolated from a full-scale bioreactor removing H(2)S from biogas under oxygen-limited conditions. Another strain, ALJ17, closely related to HL-EbGr7(T), was isolated from a Kenyan soda lake. Cells of the isolates were relatively long, slender rods, motile by a polar flagellum. Although both strains were obligately aerobic, micro-oxic conditions were preferred, especially at the beginning of growth. Chemolithoautotrophic growth was observed with sulfide and thiosulfate in a pH range of 8.0-10.5 (optimum at pH 10.0) and a salinity range of 0.2-1.5 M total Na(+) (optimum at 0.4 M). The genome sequence of strain HL-EbGr7(T) demonstrated the presence of genes encoding the reverse Dsr pathway and a truncated Sox pathway for sulfur oxidation and enzymes of the Calvin-Benson cycle of autotrophic CO(2) assimilation with ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) type I. The dominant cellular fatty acids were C(18:1)omega7, C(16:0) and C(19:0) cyclo. Based on 16S rRNA gene sequencing, the two strains belonged to a single phylotype within the genus Thioalkalivibrio in the Gammaproteobacteria. Despite being related most closely to Thioalkalivibrio denitrificans, the isolates were unable to grow by denitrification. On the basis of phenotypic and phylogenetic analysis, the novel isolates are proposed to represent a novel species, Thioalkalivibrio sulfidiphilus sp. nov., with the type strain HL-EbGr7(T) ( = NCCB 100376(T) = UNIQEM U246(T)).

• Mininno M et al.
• Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants.
• J Biol Chem. 2012; 287: 21034-44
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• In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.

• Szalaine Agoston B, Kovacs D, Tompa P, Perczel A
• Full backbone assignment and dynamics of the intrinsically disordered dehydrin ERD14.
• Biomol NMR Assign. 2011; 5: 189-93
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• Dehydrins are a class of stress proteins that belong to the family of Late Embryogenesis Abundant (LEA) proteins in plants, so named because they are highly expressed in late stages of seed formation. In somatic cells, their expression is very low under normal conditions, but increases critically upon dehydration elicited by water stress, high salinity or cold. Dehydrins are thought to be intrinsically disordered proteins, which represents a challenge in understanding their structure-function relationship. Herein we present the backbone (1)H, (15)N and (13)C NMR assignment of the 185 amino acid long ERD14 (Early Response to Dehydration 14), which is a K(3)S-type, typical dehydrin of A. thaliana. Secondary chemical shifts as well as NMR relaxation data show that ERD14 is fully disordered under near native conditions, with short regions of somewhat restricted motion and 5-25% helical propensity. These results suggest that ERD14 may have partially preformed elements for functional interaction with its partner(s) and set the stage for further detailed structural and functional studies of ERD14 both in vitro and in vivo.

• Peterson FC et al.
• Orphan macrodomain protein (human C6orf130) is an O-acyl-ADP-ribose deacylase: solution structure and catalytic properties.
• J Biol Chem. 2011; 286: 35955-65
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• Post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. Deacylation by NAD(+)-dependent sirtuin reactions yields as a product O-acyl-ADP-ribose, which has been implicated as a signaling molecule in modulating cellular processes. Macrodomain-containing proteins are reported to bind NAD(+)-derived metabolites. Here, we describe the structure and function of an orphan macrodomain protein, human C6orf130. This unique 17-kDa protein is a stand-alone macrodomain protein that occupies a distinct branch in the phylogenic tree. We demonstrate that C6orf130 catalyzes the efficient deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADPr) and acetate, propionate, and butyrate, respectively. Using NMR spectroscopy, we solved the structure of C6orf130 in the presence and absence of ADPr. The structures showed a canonical fold with a deep ligand (ADPr)-binding cleft. Structural comparisons of apo-C6orf130 and the ADPr-C6orf130 complex revealed fluctuations of the beta(5)-alpha(4) loop that covers the bound ADPr, suggesting that the beta(5)-alpha(4) loop functions as a gate to sequester substrate and offer flexibility to accommodate alternative substrates. The ADPr-C6orf130 complex identified amino acid residues involved in substrate binding and suggested residues that function in catalysis. Site-specific mutagenesis and steady-state kinetic analyses revealed two critical catalytic residues, Ser-35 and Asp-125. We propose a catalytic mechanism for deacylation of O-acyl-ADP-ribose by C6orf130 and discuss the biological implications in the context of reversible protein acylation at lysine residues.

• Qiu Q et al.
• Genome-scale transcriptome analysis of the desert poplar, Populus euphratica.
• Tree Physiol. 2011; 31: 452-61
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• Populus euphratica is well-adapted to extreme desert environments and is an important model species for studying the effects of abiotic stresses on trees. Here we present the first deep transcriptomic analysis of this species. To maximize representation of conditional transcripts, mRNA was obtained from living tissues of desert-grown trees and two types of callus (salt-stressed and unstressed). De novo assembly generated 86,777 Unigenes using Solexa sequence data. These sequences covered 92% of previously reported P. euphratica expressed sequence tags (ESTs) and 90% of the TIGR poplar ESTs, and a total of 58,499 high-quality unique sequences were annotated by BLAST similarity searches against public databases. We found that 27% of the total Unigenes were differentially expressed (up- or down-regulated) in response to salt stress in P. euphratica callus. These differentially expressed genes are mainly involved in transport, transcription, cellular communication and metabolism. In addition, we found that numerous putative genes involved in ABA regulation and biosynthesis were also differentially regulated. This study represents the deepest transcriptomic and gene-annotation analysis of P. euphratica to date. The genetic knowledge acquired should be very useful for future studies of the molecular adaptation of this tree species to abiotic stress and facilitate genetic manipulation of other poplar species.

• Herschbach C et al.
• Changes in sulphur metabolism of grey poplar (Populus x canescens) leaves during salt stress: a metabolic link to photorespiration.
• Tree Physiol. 2010; 30: 1161-73
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• The poplar hybrid Populus x canescens (syn. Populus tremula x Populus alba) was subjected to salt stress by applying 75 mM NaCl for 2 weeks in hydroponic cultures. Decreasing maximum quantum yield (Fv/Fm) indicated damage of photosystem II (PS II), which was more pronounced under nitrate compared with ammonium nutrition. In vivo staining with diaminobenzidine showed no accumulation of H(2)O(2) in the leaf lamina; moreover, staining intensity even decreased. But at the leaf margins, development of necrotic tissue was associated with a strong accumulation of H(2)O(2). Glutathione (GSH) contents increased in response to NaCl stress in leaves but not in roots, the primary site of salt exposure. The increasing leaf GSH concentrations correlated with stress-induced decreases in transpiration and net CO(2) assimilation rates at light saturation. Enhanced rates of photorespiration could also be involved in preventing reactive oxygen species formation in chloroplasts and, thus, in protecting PS II from damage. Accumulation of Gly and Ser in leaves indeed indicates increasing rates of photorespiration. Since Ser and Gly are both immediate precursors of GSH that can limit GSH synthesis, it is concluded that the salt-induced accumulation of leaf GSH results from enhanced photorespiration and is thus probably restricted to the cytosol.

• Brinker M et al.
• Linking the salt transcriptome with physiological responses of a salt-resistant Populus species as a strategy to identify genes important for stress acclimation.
• Plant Physiol. 2010; 154: 1697-709
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• To investigate early salt acclimation mechanisms in a salt-tolerant poplar species (Populus euphratica), the kinetics of molecular, metabolic, and physiological changes during a 24-h salt exposure were measured. Three distinct phases of salt stress were identified by analyses of the osmotic pressure and the shoot water potential: dehydration, salt accumulation, and osmotic restoration associated with ionic stress. The duration and intensity of these phases differed between leaves and roots. Transcriptome analysis using P. euphratica-specific microarrays revealed clusters of coexpressed genes in these phases, with only 3% overlapping salt-responsive genes in leaves and roots. Acclimation of cellular metabolism to high salt concentrations involved remodeling of amino acid and protein biosynthesis and increased expression of molecular chaperones (dehydrins, osmotin). Leaves suffered initially from dehydration, which resulted in changes in transcript levels of mitochondrial and photosynthetic genes, indicating adjustment of energy metabolism. Initially, decreases in stress-related genes were found, whereas increases occurred only when leaves had restored the osmotic balance by salt accumulation. Comparative in silico analysis of the poplar stress regulon with Arabidopsis (Arabidopsis thaliana) orthologs was used as a strategy to reduce the number of candidate genes for functional analysis. Analysis of Arabidopsis knockout lines identified a lipocalin-like gene (AtTIL) and a gene encoding a protein with previously unknown functions (AtSIS) to play roles in salt tolerance. In conclusion, by dissecting the stress transcriptome of tolerant species, novel genes important for salt endurance can be identified.

• Sun J et al.
• Calcium mediates root K+/Na+ homeostasis in poplar species differing in salt tolerance.
• Tree Physiol. 2009; 29: 1175-86
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• Using the non-invasively ion-selective microelectrode technique, flux profiles of K(+), Na(+) and H(+) in mature roots and apical regions, and the effects of Ca(2+) on ion fluxes were investigated in salt-tolerant poplar species, Populus euphratica Oliver and salt-sensitive Populus simonii x (P. pyramidalis + Salix matsudana) (Populus popularis 35-44, P. popularis). Compared to P. popularis, P. euphratica roots exhibited a greater capacity to retain K(+) after exposure to a salt shock (SS, 100 mM NaCl) and a long-term (LT) salinity (50 mM NaCl, 3 weeks). Salt shock-induced K(+) efflux in the two species was markedly restricted by K(+) channel blocker, tetraethylammonium chloride, but enhanced by sodium orthovanadate, the inhibitor of plasma membrane (PM) H(+)-ATPase, suggesting that the K(+) efflux is mediated by depolarization-activated (DA) channels, e.g., KORCs (outward rectifying K(+) channels) and NSCCs (non-selective cation channels). Populus euphratica roots were more effective to exclude Na(+) than P. popularis in an LT experiment, resulting from the Na(+)/H(+) antiport across the PM. Moreover, pharmacological evidence implies that the greater ability to control K(+)/Na(+) homeostasis in salinized P. euphratica roots is associated with the higher H(+)-pumping activity, which provides an electrochemical H(+) gradient for Na(+)/H(+) exchange and simultaneously decreases the NaCl-induced depolarization of PM, thus reducing Na(+) influx via NSCCs and K(+) efflux through DA-KORCs and DA-NSCCs. Ca(2+) application markedly limited salt-induced K(+) efflux but enhanced the apparent Na(+) efflux, thus enabling the two species, especially the salt-sensitive poplar, to retain K(+)/Na(+) homeostasis in roots exposed to prolonged NaCl treatment.

• Sun J et al.
• NaCl-induced alternations of cellular and tissue ion fluxes in roots of salt-resistant and salt-sensitive poplar species.
• Plant Physiol. 2009; 149: 1141-53
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• Using the scanning ion-selective electrode technique, fluxes of H+, Na+, and Cl- were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na+ after a short-term exposure to 50 mM NaCl (24 h) and a long term in a saline environment of 100 mM NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na+ efflux and a correspondingly increased H+ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na+/H+ exchange in root tissues and cells was inhibited by amiloride (a Na+/H+ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H+-ATPase inhibitor). These results indicate that the Na+ extrusion in stressed P. euphratica roots is the result of an active Na+/H+ antiport across the plasma membrane. In comparison, the Na+/H+ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na+ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl- exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H+, Na+, and Cl- differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.

• Ye CY, Zhang HC, Chen JH, Xia XL, Yin WL
• Molecular characterization of putative vacuolar NHX-type Na(+)/H(+) exchanger genes from the salt-resistant tree Populus euphratica.
• Physiol Plant. 2009; 137: 166-74
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• The vacuolar NHX-type Na(+)/H(+) exchangers play a key role in salt tolerance in plants. However, little is known about the Na(+)/H(+) exchangers in the salt-resistant tree, Populus euphratica. In this study, we identified six putative vacuolar Na(+)/H(+) exchanger genes from P. euphratica, designated as PeNHX1-6. Real-time polymerase chain reaction indicated that the PeNHX1/3/6 transcripts were abundant compared with the other three PeNHX genes in the three tissues (roots, stems and leaves) examined. After NaCl treatment for 6 h, the transcript levels of PeNHX1-6 were upregulated in the roots. To address the function of PeNHX1-6, complementation studies were performed with the salt-sensitive yeast mutant strain R100, which lacks activity of the endosomal Na(+)/H(+) antiporter NHX1. The results showed that PeNHX1-6 compensates, at least in part, for the function of yeast NHX1. Moreover, PeNHX3 was targeted to the tonoplast when transiently expressed in onion. Together, these results suggest that PeNHX1-6 function as vacuolar Na(+)/H(+) exchangers and that PeNHX products play an important role in the salt resistance of P. euphratica.

• Taoka K, Tsuji H, Shimamoto K
• [Structural aspects of florigen to understand the molecular mechanism of flowering].
• Tanpakushitsu Kakusan Koso. 2009; 54: 1702-7

• Diedhiou CJ, Popova OV, Golldack D
• Transcript profiling of the salt-tolerant Festuca rubra ssp. litoralis reveals a regulatory network controlling salt acclimatization.
• J Plant Physiol. 2009; 166: 697-711
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• We report an analysis of salt-stress responses in the monocotyledonous halophyte Festuca rubra ssp. litoralis. Salt-dependent expression of transcripts encoding a PIP2;1 aquaporin, V-ATPase subunit B, and the Na+/H+ antiporter NHX was characterized. Transcription of FrPIP2;1, FrVHA-B, and FrNHX1 was induced in root tissue of F. rubra ssp. litoralis by salt treatment, and during salt-stress F. rubra ssp. litoralis accumulated sodium in leaves and roots. Cell specificity of FrPIP2;1, FrVHA-B, and FrNHX1 transcription was analyzed by in situ PCR in roots of F. rubra ssp. litoralis. Expression of the genes was localized to the root epidermis, cortex cells, endodermis, and the vascular tissue. In plants treated with 500 mM NaCl, transcripts were repressed in the epidermis and the outer cortex cells, whereas endodermis and vasculature showed strong signals. These data demonstrate that transcriptional regulation of the aquaporin PIP2;1, V-ATPase, and the Na+/H+ antiporter NHX is correlated with salt tolerance in F. rubra ssp. litoralis and suggests coordinated control of ion homeostasis and water status at high salinity in plants. Salt-induced transcript accumulation in F. rubra ssp. litoralis was further monitored by cDNA-arrays with expressed sequence tags derived from a cDNA subtraction library. The salt-regulated transcripts included those involved in the control of gene expression and signal transduction elements such as a serine/threonine protein kinase, an SNF1-related protein kinase, and a WRKY-type transcription factor. Other ESTs with salt-dependent regulation included transcripts encoding proteins that function in metabolism, general stress responses, and defense and transport proteins.

• Ohyama K, Shinohara H, Ogawa-Ohnishi M, Matsubayashi Y
• A glycopeptide regulating stem cell fate in Arabidopsis thaliana.
• Nat Chem Biol. 2009; 5: 578-80
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• The secreted peptide gene CLAVATA3 (CLV3) regulates stem cell fate in the shoot apical meristem in Arabidopsis thaliana plants, but the molecular structure of the active mature CLV3 peptide is controversial. Here, using nano-LC-MS/MS analysis of apoplastic peptides of A. thaliana plants overexpressing CLV3, we show that CLV3 is a 13-amino-acid arabinosylated glycopeptide. Post-translational arabinosylation of CLV3 is critical for its biological activity and high-affinity binding to its receptor CLV1.

• Jain M, Khurana JP
• Transcript profiling reveals diverse roles of auxin-responsive genes during reproductive development and abiotic stress in rice.
• FEBS J. 2009; 276: 3148-62
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• Auxin influences growth and development in plants by altering gene expression. Many auxin-responsive genes have been characterized in Arabidopsis in detail, but not in crop plants. Earlier, we reported the identification and characterization of the members of the GH3, Aux/IAA and SAUR gene families in rice. In this study, whole genome microarray analysis of auxin-responsive genes in rice was performed, with the aim of gaining some insight into the mechanism of auxin action. A comparison of expression profiles of untreated and auxin-treated rice seedlings identified 315 probe sets representing 298 (225 upregulated and 73 downregulated) unique genes as auxin-responsive. Functional categorization revealed that genes involved in various biological processes, including metabolism, transcription, signal transduction, and transport, are regulated by auxin. The expression profiles of auxin-responsive genes identified in this study and those of the members of the GH3, Aux/IAA, SAUR and ARF gene families were analyzed during various stages of vegetative and reproductive (panicle and seed) development by employing microarray analysis. Many of these genes are, indeed, expressed in a tissue-specific or developmental stage-specific manner, and the expression profiles of some of the representative genes were confirmed by real-time PCR. The differential expression of auxin-responsive genes during various stages of panicle and seed development implies their involvement in diverse developmental processes. Moreover, several auxin-responsive genes were differentially expressed under various abiotic stress conditions, indicating crosstalk between auxin and abiotic stress signaling.

• Backman HG, Pessoa J, Eneqvist T, Glaser E
• Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP.
• FEBS Lett. 2009; 583: 2727-33
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• The dual-targeted mitochondrial and chloroplastic zinc metallooligopeptidase from Arabidopsis, AtPreP, functions as a peptidasome that degrades targeting peptides and other small unstructured peptides. In addition to Zn located in the catalytic site, AtPreP also contains two Mg-binding sites. We have investigated the role of Mg-binding using AtPreP variants, in which one or both sites were rendered unable to bind Mg(2+). Our results show that metal binding besides that of the active site is crucial for AtPreP proteolysis, particularly the inner site appears essential for normal proteolytic function. This is also supported by its evolutionary conservation among all plant species of PreP.

• Wang X, Liu Z, He Y
• Responses and tolerance to salt stress in bryophytes.
• Plant Signal Behav. 2008; 3: 516-8
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• During exposure to salt environments, plants could perceive salt signal and transmit the signal to cellular machinery to activate adaptive responses. In bryophytes, salt signal components and transcript factor identified suggest that salt activate adaptive responses to tolerate adverse environments. The ability of bryophytes to tolerate salt is determined by multiple biochemical pathways. Transmembrane transport proteins that mediate ion fluxes play a curial role in ionic and osmotic homeostasis under salt environments. Defense proteins protect cells from denaturation and degradation, as well as from oxidative damage following exposure to salt stress in bryophytes. ABA and salt stress positively affect the expression of common genes that participate in protection plant cells from injure, and ABA may be responsible for the ability to tolerate salt stress in bryophytes. In this paper, we reveal the mechanisms of salt responses and tolerance in bryophytes, and imply conservation between higher plants and bryophytes in response and tolerance to salt stress.

• Yabe T et al.
• Structural analysis of Arabidopsis CnfU protein: an iron-sulfur cluster biosynthetic scaffold in chloroplasts.
• J Mol Biol. 2008; 381: 160-73
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• CnfU, a key iron-sulfur (Fe-S) cluster biosynthetic scaffold that is required for biogenesis of ferredoxin and photosystem I in chloroplasts, consists of two tandemly repeated domains in which only the N-terminal domain contains a conserved CXXC motif. We have determined the crystal structure of the metal-free dimer of AtCnfU-V from Arabidopsis thaliana at 1.35 A resolution. The N-terminal domains of the two monomers are linked together through two intermolecular disulfide bonds between the CXXC motifs. At the dimer interface, a total of four cysteine sulfur atoms provide a Fe-S cluster assembly site surrounded by uncharged but hydrophilic structurally mobile segments. The C-terminal domain of one monomer interacts with the N-terminal domain of the opposing monomer and thereby stabilizes dimer formation. Furthermore, Fe K-edge X-ray absorption spectroscopic analysis of the holo-CnfU dimer in solution suggests the presence of a typical [2Fe-2S]-type cluster coordinated by four thiolate ligands. Based on these data, a plausible model of the holo-AtCnfU-V dimer containing a surface-exposed [2Fe-2S] cluster assembled in the dimer interface was deduced. We propose that such a structural framework is important for CnfU to function as a Fe-S cluster biosynthetic scaffold.

• Lv PP et al.
• [Function of the putative Na+/H+ antiporter gene PeNhaD1 from salt-resistant Populus euphratica Oliv].
• Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2007; 33: 173-8
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• Yeast complementation experiments were carried out to define the possible function of PeNhaD1, a Na(+)/H(+) antiporter gene from Populus euphratica Oliv., a salt resistant tree. PeNhaD1 was introduced to the Saccharomyces cerevisiae mutant strain ANT3 (Deltaena1-4::HIS3 Deltanha1::LEU2), which lacks the plasma membrane Na(+)/H(+) antiporter gene ScNHA1 or GX1 (Deltanhx1::TRP1), which lacks tonoplast Na(+)/H(+) antiporter gene ScNHX1. Our results showed that PeNhaD1 rescued the normal growth of ANT3 in the presence of high salt (80 mmol/L NaCl on solid medium or 400 mmol/L in liquid medium, pH 6.0), but not that of GX1, suggesting that PeNhaD1 may play a role in salt tolerance of Populus euphratica by maintaining the capacity for salt exclusion under saline condition.

• Zhang F, Wang Y, Yang Y, Wu H, Wang D, Liu J
• Involvement of hydrogen peroxide and nitric oxide in salt resistance in the calluses from Populus euphratica.
• Plant Cell Environ. 2007; 30: 775-85
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• Nitric oxide (NO) and hydrogen peroxide (H2O2) function as signalling molecules in plants under abiotic and biotic stresses. Calluses from Populus euphratica, which show salt tolerance, were used to study the interaction of NO and H2O2 in plant adaptation to salt resistance. The nitric oxide synthase (NOS) activity was identified in the calluses, and this activity was induced under 150 mM NaCl treatment. Under 150 mM NaCl treatment, the sodium (Na) percentage decreased, but the potassium (K) percentage and the K/Na ratio increased in P. euphratica calluses. Application of glucose/glucose oxidase (G/GO, a H2O2 donor) and sodium nitroprusside (SNP, a NO donor) revealed that both H2O2 and NO resulted in increased K/Na ratio in a concentration-dependent manner. Diphenylene iodonium (DPI, an NADPH oxidase inhibitor) counteracted H2O2 and NO effect by increasing the Na percentage, decreasing the K percentage and K/Na ratio. NG-monomethyl-L-Arg monoacetate (NMMA, an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO, a specific NO scavenger) only reversed NO effect, but did not block H2O2 effect. The increased activity of plasma membrane (PM) H+ -ATPase caused by salt stress was reversed by treatment with DPI and NMMA. Exogenous H2O2 increased the activity of PM H+ -ATPase, but the effect could not be diminished by NMMA and PTIO. The NO-induced increase of PM H+ -ATPase can be reversed by NMMA and PTIO, but not by DPI. Western blot analysis demonstrated that NO and H2O2 stimulated the expression of PM H+ -ATPase in P. euphratica calluses. These results indicate that NO and H2O2 served as intermediate molecules in inducing salt resistance in the calluses from P. euphratica under slat stress by increasing the K/Na ratio, which was dependent on the increased PM H+ -ATPase activity.

• Qin F et al.
• Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L.
• Plant J. 2007; 50: 54-69
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• DREB1/CBFs and DREB2s are transcription factors that specifically interact with a cis-acting element, DRE/CRT, which is involved in the expression of genes responsive to cold and drought stress in Arabidopsis thaliana. The function of DREB1/CBFs has been precisely analyzed and it has been found to activate the expression of many genes responsive to cold stress containing a DRE/CRT sequence in their promoters. However, the regulation and function of DREB2-type transcription factors remained to be elucidated. In this research, we report the cloning of a DREB2 homolog from maize, ZmDREB2A, whose transcripts were accumulated by cold, dehydration, salt and heat stresses in maize seedlings. Unlike Arabidopsis DREB2A, ZmDREB2A produced two forms of transcripts, and quantitative real-time PCR analyses demonstrated that only the functional transcription form of ZmDREB2A was significantly induced by stresses. Moreover, the ZmDREB2A protein exhibited considerably high transactivation activity compared with DREB2A in Arabidopsis protoplasts, suggesting that protein modification is not necessary for ZmDREB2A to be active. Constitutive or stress-inducible expression of ZmDREB2A resulted in an improved drought stress tolerance in plants. Microarray analyses of transgenic plants overexpressing ZmDREB2A revealed that in addition to genes encoding late embryogenesis abundant (LEA) proteins, some genes related to heat shock and detoxification were also upregulated. Furthermore, overexpression of ZmDREB2A also enhanced thermotolerance in transgenic plants, implying that ZmDREB2A may play a dual functional role in mediating the expression of genes responsive to both water stress and heat stress.

• Wojtas M et al.
• Cloning and characterization of Rab Escort Protein (REP) from Arabidopsis thaliana.
• Cell Biol Int. 2007; 31: 246-51
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• Intracellular vesicular trafficking is regulated by Rab proteins, small GTPases that require posttranslational geranylgeranylation for biological activity. This covalent modification is catalyzed by Rab geranylgeranyl transferase (RabGGTase) and proceeds only in the presence of accessory Rab Escort Protein (REP). In this communication, we report the cloning and characterization of REP gene of Arabidopsis thaliana. Highest expression of REP mRNA was detected in leaves and flowers in contrast to stems and roots. AtREP is recognized by anti-rat REP1 serum. Interaction of AtREP with the protein substrate is presented, as well as a structural model obtained through homology modeling, based on the known structure of rat REP1.

• Langenfeld-Heyser R et al.
• Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populusxcanescens.
• Mycorrhiza. 2007; 17: 121-31
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• In order to characterise the effect of ectomycorrhiza on Na+-responses of the salt-sensitive poplar hybrid Populus x canescens, growth and stress responses of Paxillus involutus (strain MAJ) were tested in liquid cultures in the presence of 20 to 500 mM NaCl, and the effects of mycorrhization on mineral nutrient accumulation and oxidative stress were characterised in mycorrhizal and non-mycorrhizal poplar seedlings exposed to 150 mM NaCl. Paxillus involutus was salt tolerant, showing biomass increases in media containing up to 500 mM NaCl after 4 weeks growth. Mycorrhizal mantle formation on poplar roots was not affected by 150 mM NaCl. Whole plant performance was positively affected by the fungus because total biomass was greater and leaves accumulated less Na+ than non-mycorrhizal plants. Energy dispersive X-ray microanalysis using transmission electron microscopy analysis of the influence of mycorrhization on the subcellular localisation of Na+ and Cl- in roots showed that the hyphal mantle did not diminish salt accumulation in root cell walls, indicating that mycorrhization did not provide a physical barrier against excess salinity. In the absence of salt stress, mycorrhizal poplar roots contained higher Na+ and Cl- concentrations than non-mycorrhizal poplar roots. Paxillus involutus hyphae produced H2O2 in the mantle but not in the Hartig net or in pure culture. Salt exposure resulted in H2O2 formation in cortical cells of both non-mycorrhizal and mycorrhizal poplar and stimulated peroxidase but not superoxide dismutase activities. This shows that mature ectomycorrhiza was unable to suppress salt-induced oxidative stress. Element analyses suggest that improved performance of mycorrhizal poplar under salt stress may result from diminished xylem loading of Na+ and increased supply with K+.

• Ouyang B et al.
• Identification of early salt stress response genes in tomato root by suppression subtractive hybridization and microarray analysis.
• J Exp Bot. 2007; 58: 507-20
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• High salinity is one of the most serious threats to crop production. To understand the molecular basis of plant responses to salt stress better, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes involved in the early stage of tomato responses to severe salt stress. First, SSH libraries were constructed for the root tissue of two cultivated tomato (Solanum lycopersicum) genotypes: LA2711, a salt-tolerant cultivar, and ZS-5, a salt-sensitive cultivar, to compare salt treatment and non-treatment plants. Then a subset of clones from these SSH libraries were used to construct a tomato cDNA array and microarray analysis was carried out to verify the expression changes of this set of clones upon a high concentration of salt treatment at various time points compared to the corresponding non-treatment controls. A total of 201 non-redundant genes that were differentially expressed upon 30 min of severe salt stress either in LA2711 or ZS-5 were identified from microarray analysis; most of these genes have not previously been reported to be associated with salt stress. The diversity of the putative functions of these genes indicated that salt stress resulted in a complex response in tomato plants.

• Eren E, Gonzalez-Guerrero M, Kaufman BM, Arguello JM
• Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2.
• Biochemistry. 2007; 46: 7754-64
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• Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.

• de Maria N et al.
• Putative porin of Bradyrhizobium sp. (Lupinus) bacteroids induced by glyphosate.
• Appl Environ Microbiol. 2007; 73: 5075-82
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• Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed.

• Zhang F, Wang Y, Wang D
• Role of nitric oxide and hydrogen peroxide during the salt resistance response.
• Plant Signal Behav. 2007; 2: 473-4
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• Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H(2)O(2) causes salt resistance via the action of plasma membrane H(+)-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H(2)O(2) mediating salt resistance.

• Lermontova I, Schubert V, Bornke F, Macas J, Schubert I
• Arabidopsis CBF5 interacts with the H/ACA snoRNP assembly factor NAF1.
• Plant Mol Biol. 2007; 65: 615-26
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• The conserved protein CBF5, initially regarded as a centromere binding protein in yeast and higher plants, was later found within nucleoli and in Cajal bodies of yeast and metazoa. There, it is assumed to be involved in posttranscriptional pseudouridinylation of various RNA species that might be important for RNA processing. We found EYFP-labeled CBF5 of A. thaliana to be located within nucleoli and Cajal bodies, but neither at centromeres nor somewhere else on chromosomes. Arabidopsis mutants carrying a homozygous T-DNA insertion at the CBF5 locus were lethal. Yeast two-hybrid and mRNA expression analyses demonstrated that AtCBF5 is co-expressed and interacts with a previously uncharacterized protein containing a conserved NAF1 domain, presumably involved in H/ACA box snoRNP biogenesis. The homologous yeast protein has been shown to contribute to RNA pseudouridinylation. Thus, AtCBF5 might have an essential function in RNA processing rather than being a kinetochore protein.

• Milla MA, Townsend J, Chang IF, Cushman JC
• The Arabidopsis AtDi19 gene family encodes a novel type of Cys2/His2 zinc-finger protein implicated in ABA-independent dehydration, high-salinity stress and light signaling pathways.
• Plant Mol Biol. 2006; 61: 13-30
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• The AtDi19 (drought-induced) gene family encodes seven hydrophilic proteins that contain two atypical Cys2/His2 (C2H2) zinc finger-like domains that are evolutionarily well-conserved within angiosperms suggesting a conserved and important function. Five of the seven Arabidopsis AtDi19-related:DsRed2 fusion proteins exhibited speckled patterns of localization within the nucleus as shown by transient expression analysis in Arabidopsis protoplasts. In contrast, AtDi19-2:DsRed2 was present in the nucleus and cytoplasm, whereas AtDi19-4:DsRed2 was localized to the nuclear periphery. mRNA expression studies showed that AtDi19 genes are ubiquitously expressed in Arabidopsis tissues, although some differences were observed. In seedlings, RT-PCR analyses showed that AtDi19-1 and AtDi19-3 steady-state transcript amounts were rapidly induced by dehydration, whereas transcript amounts for AtDi19-2 and AtDi19-4 increased in response to high-salt stress. In addition, the mRNA abundance of all the AtDi19-related gene family members was not regulated by ABA. These data, taken together, suggest that several AtDi19-related gene family members may function in ABA-independent, dehydration and salinity stress signaling pathways. However, they may also be regulated by other abiotic stimuli. AtDi19-7, for example, has been implicated in regulating light signaling and responses. Finally, we show that most AtDi19-related proteins are phosphorylated in vitro by calcium-dependent protein kinases suggesting that this post-translational modification may be important for regulating the function of this novel protein family.

• Grasser M, Lentz A, Lichota J, Merkle T, Grasser KD
• The Arabidopsis genome encodes structurally and functionally diverse HMGB-type proteins.
• J Mol Biol. 2006; 358: 654-64
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• The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription and recombination. In addition to the previously identified HMGB1-HMGB6 proteins, the Arabidopsis genome encodes at least two other candidate family members (encoded by the loci At2g34450 and At5g23405) having the typical overall structure of a central domain displaying sequence similarity to HMG-box DNA binding domains, which is flanked by basic N-terminal and acidic C-terminal regions. Subcellular localisation experiments demonstrate that the At2g34450 protein is a nuclear protein, whereas the At5g23405 protein is found mainly in the cytoplasm. In line with this finding, At5g23405 displays specific interaction with the nuclear export receptor AtXPO1a. According to CD measurements, the HMG-box domains of both proteins have an alpha-helical structure. The HMG-box domain of At2g34450 interacts with linear DNA and binds structure-specifically to DNA minicircles, whereas the HMG-box domain of At5g23405 does not interact with DNA at all. In ligation experiments with short DNA fragments, the At2g34450 HMG-box domain can facilitate the formation of linear oligomers, but it does not promote the formation of DNA minicircles. Therefore, the At2g34450 protein shares several features with HMGB proteins, whereas the At5g23405 protein has different characteristics. Despite the presence of a region with similarity to the nucleosome-binding domain typical of HMGN proteins, At2g34450 does not bind nucleosome particles. In summary, our data demonstrate (i) that plant HMGB-type proteins are functionally variable and (ii) that it is difficult to predict HMG-box function solely based on sequence similarity.

• Ben-Naim O et al.
• The CCAAT binding factor can mediate interactions between CONSTANS-like proteins and DNA.
• Plant J. 2006; 46: 462-76
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• CONSTANS-Like (COL) proteins are plant-specific nuclear regulators of gene expression but do not contain a known DNA-binding motif. We tested whether a common DNA-binding protein can deliver these proteins to specific cis-acting elements. We screened for proteins that interact with two members of a subgroup of COL proteins. These COL proteins were Tomato COL1 (TCOL1), which does not seem to be involved in the control of flowering time, and the Arabidopsis thaliana CONSTANS (AtCO) protein which mediates photoperiodic induction of flowering. We show that the C-terminal plant-specific CCT (CO, CO-like, TIMING OF CAB EXPRESSION 1) domain of both proteins binds the trimeric CCAAT binding factor (CBF) via its HAP5/NF-YC component. Chromatin immunoprecipitation demonstrated that TCOL is recruited to the CCAAT motifs of the yeast CYC1 and HEM1 promoters by HAP5. In Arabidopsis, each of the three CBF components is encoded by several different genes that are highly transcribed. Under warm long days, high levels of expression of a tomato HAP5 (THAP5a) gene can reduce the flowering time of Arabidopsis. A mutation in the CCT domain of TCOL1 disrupts the interaction with THAP5 and the analogous mutation in AtCO impairs its function and delays flowering. CBFs are therefore likely to recruit COL proteins to their DNA target motifs in planta.

• Sue SC et al.
• Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix.
• J Mol Biol. 2006; 356: 72-85
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• The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.

• Kader MA, Seidel T, Golldack D, Lindberg S
• Expressions of OsHKT1, OsHKT2, and OsVHA are differentially regulated under NaCl stress in salt-sensitive and salt-tolerant rice (Oryza sativa L.) cultivars.
• J Exp Bot. 2006; 57: 4257-68
• Display abstract

• Under NaCl-dominated salt stress, the key to plant survival is maintaining a low cytosolic Na(+) level or Na(+)/K(+) ratio. The OsHKT1, OsHKT2, and OsVHA transporter genes might play important roles in maintaining cytosolic Na(+) homeostasis in rice (Oryza sativa L. indica cvs Pokkali and BRRI Dhan29). Upon NaCl stress, the OsHKT1 transcript was significantly down-regulated in salt-tolerant cv. Pokkali, but not in salt-sensitive cv. BRRI Dhan29. NaCl stress induced the expression of OsHKT2 and OsVHA in both Pokkali and BRRI Dhan29. In cv. Pokkali, OsHKT2 and OsVHA transcripts were induced immediately after NaCl stress. However, in cv. BRRI Dhan29, the induction of OsHKT2 was quite low and of OsVHA was low and delayed, compared with that in cv. Pokkali. OsHKT2 and OsVHA induction mostly occurred in the phloem, in the transition from phloem to mesophyll cells, and in the mesophyll cells of the leaves. The vacuolar area in cv. Pokkali did not change under either short- (5-10 min) or long-term (24 h) salt stress, although it significantly increased 24 h after the stress in cv. BRRI Dhan29. When expressional constructs of VHA-c and VHA-a with YFP and CFP were introduced into isolated protoplasts of cvs Pokkali and BRRI Dhan29, the fluorescence resonance energy transfer (FRET) efficiency between VHA-c and VHA-a upon salt stress decreased slightly in cv. Pokkali, but increased significantly in cv. BRRI Dhan29. The results suggest that the salt-tolerant cv. Pokkali regulates the expression of OsHKT1, OsHKT2, and OsVHA differently from how the salt-sensitive cv. BRRI Dhan29 does. Together, these proteins might confer salt tolerance in Pokkali by maintaining a low cytosolic Na(+) level and a correct ratio of cytosolic Na(+)/K(+).

• Jithesh MN, Prashanth SR, Sivaprakash KR, Parida A
• Monitoring expression profiles of antioxidant genes to salinity, iron, oxidative, light and hyperosmotic stresses in the highly salt tolerant grey mangrove, Avicennia marina (Forsk.) Vierh. by mRNA analysis.
• Plant Cell Rep. 2006; 25: 865-76
• Display abstract

• Plant photosynthesis results in the production of molecular oxygen. An inevitable consequence of this normal process is the production of reactive oxygen species (ROS) by the transfer of electrons to molecular oxygen. Plants are adequately protected by the presence of multiple antioxidative enzymes in different organelles of the plant such as chloroplasts, cytosol, mitochondria and peroxisomes. Under high light and CO(2) limiting conditions caused by environmental stress like salinity, these antioxidative enzymes play an important role in scavenging toxic radicals. To investigate the functions of antioxidative enzymes in a mangrove plant, we isolated three cDNAs encoding cytosolic Cu-Zn SOD (Sod1), catalase (Cat1) and ferritin (Fer1) from Avicennia marina cDNA library. Sod1, Cat1 and Fer1 cDNA encoded full-length proteins with 152, 492 and 261 amino acids respectively. We studied the expression of these antioxidant genes in response to salt, iron, hydrogen peroxide, mannitol and light stress by mRNA expression analysis. Cat1, Fer1 showed short-term induction while Sod1 transcript was found to be unaltered in response to NaCl stress. A decrease in mRNA levels was observed for Sod1, Cat1 while Fer1 mRNA levels remained unaltered with osmotic stress treatment. Sod1, Cat1 and Fer1 mRNA levels were induced by iron, light stress and by direct H(2)O(2) stress treatment, thus confirming their role in oxidative stress response.

• Nakasako M, Matsuoka D, Zikihara K, Tokutomi S
• Quaternary structure of LOV-domain containing polypeptide of Arabidopsis FKF1 protein.
• FEBS Lett. 2005; 579: 1067-71
• Display abstract

• Flavin-binding, Kelch repeat, F-box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)-sensing domain binding a flavin mononucleotide. The photo-activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV-containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small-angle X-ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic-shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27 A and the maximum dimension of 94 A. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue-light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino-acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.

• Abdel-Ghany SE et al.
• AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7.
• FEBS Lett. 2005; 579: 2307-12
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• In plant chloroplasts two superoxide dismutase (SOD) activities occur, FeSOD and Cu/ZnSOD, with reciprocal regulation in response to copper availability. This system presents a unique model to study the regulation of metal-cofactor delivery to an organelle. The Arabidopsis thaliana gene AtCCS encodes a functional homolog to yeast Ccs1p/Lys7p, a copper chaperone for SOD. The AtCCS protein was localized to chloroplasts where it may supply copper to the stromal Cu/ZnSOD. AtCCS mRNA expression levels are upregulated in response to Cu-feeding and senescence. We propose that AtCCS expression is regulated to allow the most optimal use of Cu for photosynthesis.

• Yamasaki K et al.
• Solution structure of the major DNA-binding domain of Arabidopsis thaliana ethylene-insensitive3-like3.
• J Mol Biol. 2005; 348: 253-64
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• Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the ethylene signaling of higher plants. The EIN3/EIL proteins bind to the promoter regions of the downstream genes and regulate their expression. The location of the DNA-binding domain (DBD) in the primary structure was unclear, since the proteins show no sequence similarity to other known DBDs. Here, we identify the major DBD of an EIN3/EIL protein, Arabidopsis thaliana EIL3, containing a key mutational site for DNA binding and signaling (ein3-3 site), and determine its solution structure by NMR spectroscopy. The structure consists of five alpha-helices, possessing a novel fold dissimilar to known DBD structures. By a chemical-shift perturbation analysis, a region including the ein3-3 site is suggested to be involved in DNA binding.

• Song J, Zhao Q, Lee MS, Markley JL
• 1H, 15N and 13C resonance assignments of the putative Bet v 1 family protein At1g24000.1 from Arabidopsis thaliana.
• J Biomol NMR. 2005; 32: 335-335

• Turova TP, Spiridonova EM, Berg IA, Kuznetsov BB, Sorokin DIu
• [Phylogeny of ribulose-1,5-bisphosphate carboxylase/oxygenase genes in haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio].
• Mikrobiologiia. 2005; 74: 378-86
• Display abstract

• Fragments of genes of the greenlike form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of eight species of haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio have been revealed and sequenced using previously developed oligonucleotide primers. The data obtained are used for the construction of phylogenetic trees on the basis of nucleotide sequences of RuBisCO genes and their conceptual translations into amino acid sequences. Comparative analysis of the 16S rRNA and RuBisCO gene trees reveals discrepancies between their topologies. According to a RuBisCO gene analysis, the genus Thioalkalivibrio is not monophyletic, and its inner divergence conforms to the significant morphological differences observed between the species. Presumably, horizontal (interspecies) gene transfer was involved in the evolution of the genus Thioalkalivibrio.

• Wang JY, Yin WL, Xia XL
• [Cloning and structure analysis of zinc finger protein gene in Populus euphratica Oliv].
• Yi Chuan. 2005; 27: 245-8
• Display abstract

• Zinc finger proteins belong to a family of nuclear transcription factors which function is to regulate gene expression in both prokaryotic and eukaryotic cells. A pair of primers was designed after analyzing the conservation of salt-tolerant zinc protein Alfin-1 in such diverse plants as alfalfa and Arabidopsis. The zinc finger protein gene is isolated from total RNA with RT-PCR in aquaculture leaves of Populus euphratica . Its full cDNA length is 924bp. Analysis of its amino acid sequence showed it has a typical Cys(2)/His(2) zinc finger structure and a G-rich promoter binding site GTGGGG, starting from position 556. Since transcrptional factors which have the same function show conservation in structure and amino acid sequence of DNA binding region, the structure analysis in this paper indicates the cloned zinc finger protein gene may have functional correlation to Alfin-1.

• Yamasaki K et al.
• A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP-family transcription factors.
• J Mol Biol. 2004; 337: 49-63
• Display abstract

• SQUAMOSA promoter binding proteins (SBPs) form a major family of plant-specific transcription factors related to flower development. Although SBPs are heterogeneous in primary structure, they share a highly conserved DNA-binding domain (DBD) that has been suggested to be zinc binding. Here we report the NMR solution structures of DBDs of two SBPs of Arabidopsis thaliana, SPL4 and SPL7. The two share essentially the same structural features. Each structure contains two zinc-binding sites consisting of eight Cys or His residues in a Cys3HisCys2HisCys or Cys6HisCys sequence motif in which the first four residues coordinate to one zinc and the last four coordinate to the other. These structures are dissimilar to other known zinc-binding structures, and thus represent a novel type of zinc-binding motif. The electrostatic profile on the surface suggested that a continuous region, including all the conserved basic residues, is involved in the DNA binding, the mode of which is likely to be novel as well.

• Im YJ et al.
• Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling.
• J Mol Biol. 2004; 343: 659-70
• Display abstract

• In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.

• Lopez-Mendez B et al.
• NMR assignment of the hypothetical ENTH-VHS domain At3g16270 from Arabidopsis thaliana.
• J Biomol NMR. 2004; 29: 205-6

• Willard FS, Siderovski DP
• Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G-protein signaling-1.
• Methods Enzymol. 2004; 389: 320-38
• Display abstract

• The model organism Arabidopsis thaliana contains a restricted set of heterotrimeric G-protein subunits, with only one canonical Galpha subunit (AtGPA1), one Gbeta subunit (AtAGB1), and two Ggamma subunits (AtAGG1 and AtGG2) identified. We have identified a novel additional component of heterotrimeric G-protein signaling in the A. thaliana genome, regulator of G-protein signaling-1 (AtRGS1). This protein has the predicted topology and structure of a G-protein-coupled receptor in that it contains seven transmembrane domains, but AtRGS1 also contains a unique C-terminal extension, namely a regulator of G-protein signaling domain (RGS box). This article describes methods for the purification and in vitro functional analysis of the RGS box of AtRGS1.

• Ueda A et al.
• Osmotic stress in barley regulates expression of a different set of genes than salt stress does.
• J Exp Bot. 2004; 55: 2213-8
• Display abstract

• Under high salt conditions, plant growth is severely inhibited due to both osmotic and ionic stresses. In an effort to dissect genes and pathways that respond to changes in osmotic potential under salt stress, the expression patterns were compared of 460 non-redundant salt-responsive genes in barley during the initial phase under osmotic versus salt stress using cDNA microarrays with northern blot and real-time RT-PCR analyses. Out of 52 genes that were differentially expressed under osmotic stress, 11, such as the up-regulated genes for pyrroline-5-carboxylate synthetase, betaine aldehyde dehydrogenase 2, plasma membrane protein 3, and the down-regulated genes for water channel 2, heat shock protein 70, and phospholipase C, were regulated in a virtually identical manner under salt stress. These genes were involved in a wide range of metabolic and signalling pathways suggesting that, during the initial phase under salt stress, several of the cellular responses are mediated by changes in osmotic potential.

• Sugimori N, Torizawa T, Aceti DJ, Thao S, Markley JL, Kainosho M
• (1)H, (13)C and (15)N backbone assignment of a 32 kDa hypothetical protein from Arabidopsis thaliana, At3g16450.1.
• J Biomol NMR. 2004; 30: 357-8

• Cort JR, Chiang Y, Zheng D, Montelione GT, Kennedy MA
• NMR structure of conserved eukaryotic protein ZK652.3 from C. elegans: a ubiquitin-like fold.
• Proteins. 2002; 48: 733-6

• Ueda A, Shi W, Nakamura T, Takabe T
• Analysis of salt-inducible genes in barley roots by differential display.
• J Plant Res. 2002; 115: 119-30
• Display abstract

• To obtain insight into the comprehensive molecular characteristics related to the mechanisms of salt tolerance, we performed a large-scale screening of salt-inducible genes in barley roots by differential display. A comparative analysis of gene expression between control and salt-stressed conditions led to the detection of 218 cDNA clones induced by salt. Sequence analysis and database searching revealed that 133 cDNA clones have homology to known proteins. Twenty-four salt-inducible clones were identified as genes for signal transduction (e.g., phosphatidylinositol-4-phosphate-5-kinase, mitogen-activated protein kinase, transcription factor, receptor protein kinase, and protein phosphatase 2A). We also detected clones encoding glutathione reductase, thioredoxin-like protein, trehalose-6-phosphate synthetase, and heat shock proteins in the category of typical stress tolerance. Furthermore, we have obtained genes encoding membrane transporters, members of the P450 family, enzymes involved in RNA metabolism or function, and enzymes of sugar or amino acid metabolism. It must be noted that most genes were expressed strongly in roots, but only rarely or weakly in leaves. In addition, some clones were newly found as salt-inducible genes encoding SCARECROW, splicing factor and apoptosis protein. In this research, it was shown that differential display is a powerful tool for a large-scale cloning of cDNAs induced by salt and these results are very useful for understanding the mechanisms of plant salt tolerance.

• Niimi T et al.
• NMR structure of human fibronectin EDA.
• J Biomol NMR. 2001; 21: 281-4

• Landrieu I, Wieruszeski JM, Odaert B, Inze D, Grzesiek S, Lippen G
• Letter to the editor: sequence-specific 1H, 13C and 15N chemical shift backbone NMR assignment and secondary structure of the Arabidopsis thaliana PIN1At protein.
• J Biomol NMR. 2000; 17: 271-2

• Ezaki S, Maeda N, Kishimoto T, Atomi H, Imanaka T
• Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1.
• J Biol Chem. 1999; 274: 5078-82
• Display abstract

• We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos. Rubisco of P. kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type. Active site residues identified from type I and type II Rubiscos were conserved. We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence. Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer. Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90 degreesC, both higher than any previously characterized Rubisco. The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degreesC. Northern blot analysis demonstrated that the gene was transcribed in P. kodakaraensis KOD1. Furthermore, Western blot analysis with cell-free extract of P. kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells.

• Sugawara H et al.
• Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita.
• J Biol Chem. 1999; 274: 15655-61
• Display abstract

• Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1. 39) obtained from a thermophilic red alga Galdieria partita has the highest specificity factor of 238 among the Rubiscos hitherto reported. Crystal structure of activated Rubisco from G. partita complexed with the reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) has been determined at 2.4-A resolution. Compared with other Rubiscos, different amino residues bring the structural differences in active site, which are marked around the binding sites of P-2 phosphate of 2-CABP. Especially, side chains of His-327 and Arg-295 show the significant differences from those of spinach Rubisco. Moreover, the side chains of Asn-123 and His-294 which are reported to bind the substrate, ribulose 1,5-bisphosphate, form hydrogen bonds characteristic of Galdieria Rubisco. Small subunits of Galdieria Rubisco have more than 30 extra amino acid residues on the C terminus, which make up a hairpin-loop structure to form many interactions with the neighboring small subunits. When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of insertion residues 52-63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other.

• Hayashi NR, Arai H, Kodama T, Igarashi Y
• The novel genes, cbbQ and cbbO, located downstream from the RubisCO genes of Pseudomonas hydrogenothermophila, affect the conformational states and activity of RubisCO.
• Biochem Biophys Res Commun. 1997; 241: 565-9
• Display abstract

• The cbbQ and cbbO genes are located downstream from the RubisCO genes (cbbLS) in the thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila. Recombinant RubisCO enzymes were purified from E. coli cells which were transformed with plasmids expressing cbbLS, cbbLSQ, cbbLSO, or cbbLSQO. Co-expression of cbbQ and/or cbbO with cbbLS made the maximal rates of carboxylation (Vmax) of the recombinant RubisCOs about two-fold higher than that of the enzyme derived from only cbbLS. The RubisCOs with high Vmax also had a high stability when undergoing ultrasonic treatment. The results of the circular dichroism spectra and the 8-anilino-1-naphthalenesulfonate binding assay indicated that these recombinant RubisCOs were conformationally different to each other.

• Kuba M et al.
• Molecular evolution of amphioxus fructose-1,6-bisphosphate aldolase.
• Arch Biochem Biophys. 1997; 348: 329-36
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• The cDNA for amphioxus fructose-1,6-bisphosphate (FBP)-aldolase was isolated and its nucleotide sequence was determined. In the cDNA, there existed a probable open reading frame comprising 1080 bp; hence, 359 amino acid residues were deduced. The amino acid sequence indicates the deletion of 4 residues from N-terminus, in comparison with the sequence of FBP-aldolase isozymes from other sources. There was only one FBP-aldolase gene, and one enzyme species corresponding, in the amphioxus; this is the first report of the existence of a single FBP-aldolase species in animals. Enzymatic studies of both native and the recombinant FBP-aldolase suggest that the amphioxus enzyme belongs to an ancestral class I type which is not discovered among vertebrate aldolase isozymes.

• Watson GM, Tabita FR
• Regulation, unique gene organization, and unusual primary structure of carbon fixation genes from a marine phycoerythrin-containing cyanobacterium.
• Plant Mol Biol. 1996; 32: 1103-15
• Display abstract

• Marine phycoerythrin-containing cyanobacteria are major contributors to the overall productivity of the oceans. The present study indicates that the structural genes of the carbon assimilatory system are unusually arranged and possess a unique primary structure compared to previously studied cyanobacteria. Southern blot analyses of Synechococcus sp. strain WH7803 chromosomal DNA digests, using the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit gene from Synechococcus sp. strain PCC6301 as a heterologous probe, revealed the presence of a 6.4 kb HindIII fragment that was detectable at only low stringency. Three complete open reading frames (ORFs) were detected within this fragment. Two of these ORFs potentially encode the Synechococcus sp. strain WH7803 rbcL and rbcS genes. The third ORF, situated immediately upstream from rbcL, potentially encodes a homologue of the ccmK gene from Synechococcus sp. strain PCC7942. The deduced amino acid sequences of each of these ORFs are more similar to homologues among the beta/gamma purple bacteria than to existing cyanobacterial homologues and phylogenetic analysis of the Rubisco large and small subunit sequences confirmed an unexpected relationship to sequences from among the beta/gamma purple bacteria. This is the first instance in which the possibility has been considered that an operon encoding three genes involved in carbon fixation may have been laterally transferred from a purple bacterium. Analysis of mRNA extracted from cells grown under diel conditions indicated that rbcL, rbcS and ccmK were regulated at the transcriptional level; specifically Rubisco transcripts were highest during the midday period, decreased at later times during the light period and eventually reached a level where they were all but undetectable during the dark period. Primer extension analysis indicated that the ccmK, rbcL and rbcS genes were co-transcribed.

• Yokoyama K, Hayashi NR, Arai H, Chung SY, Igarashi Y, Kodama T
• Genes encoding RubisCO in Pseudomonas hydrogenothermophila are followed by a novel cbbQ gene similar to nirQ of the denitrification gene cluster from Pseudomonas species.
• Gene. 1995; 153: 75-9
• Display abstract

• The cbbL and cbbS genes, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), were cloned and sequenced from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The cbbL gene encoded a 474-amino-acid (aa) protein (53,285 Da); cbbS encoded a 124-aa protein (14,656 Da). An ORF found downstream from the cbbLS genes encoded a 267-aa protein (29,565 Da) which had no similarity to cbbX located downstream from cbbLS from Alcaligenes eutrophus and Xanthobacter flavus. This gene, called cbbQ, was highly similar to the nirQ gene of the denitrification gene cluster from P. aeruginosa and P. stutzeri.

• Witke C, Gotz F
• Cloning, sequencing, and characterization of the gene encoding the class I fructose-1,6-bisphosphate aldolase of Staphylococcus carnosus.
• J Bacteriol. 1993; 175: 7495-9
• Display abstract

• fda from Staphylococcus carnosus TM300, encoding the class I fructose-1,6-bisphosphate aldolase, was cloned in Escherichia coli and sequenced. The 888-nucleotide open reading frame encoding a protein with an M(r) of 32,855 had an E. coli-like promoter sequence. Plasmids containing fda complemented E. coli NP315 (Fda-). Expression of fda in S. carnosus led to a six- to eightfold increase in aldolase production and activity; low levels of glucose in the growth medium stimulated activity.

• English RS, Williams CA, Lorbach SC, Shively JM
• Two forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Thiobacillus denitrificans.
• FEMS Microbiol Lett. 1992; 73: 111-9
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• The autotrophic, sulfur-oxidizing bacterium Thiobacillus denitrificans possesses two forms of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The form I and form II genes were isolated from a cosmid library using heterologous DNA probes. Restriction enzyme analysis indicated that the genes are within 17 kbp of each other. Other Calvin cycle enzyme genes are not present. Analysis of T. denitrificans RNA indicated that the form I genes for the large and small subunits are co-transcribed with a length of 2800 nucleotides. The transcript for the form II gene is 1900 nucleotides in length.

• Pulgar V, Gaete L, Allende J, Orellana O, Jordana X, Jedlicki E
• Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase.
• FEBS Lett. 1991; 292: 85-9
• Display abstract

• The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.

• Kobayashi H et al.
• Sequence and expression of genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chromatium vinosum.
• Gene. 1991; 97: 55-62
• Display abstract

• A DNA fragment bearing genes for the large (rbcL) and small (rbcS) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was cloned from the photosynthetic purple sulfur bacterium Chromatium vinosum. Enzymatically fully active RuBisCO was synthesized in Escherichia coli cells when the cloned DNA was placed downstream of tac promoter. Nucleotide (nt) sequences of rbcL-rbcS were more homologous to cyanobacterial counterparts than to those from Alcaligenes eutrophus or higher plants. However, the amino acid (aa) sequence in a domain responsible for CO2 activation in the C. vinosum rbcL product resembled the corresponding aa sequence in higher plant RuBisCos, but not in the cyanobacterial enzymes. Chemically determined aa sequences at the N terminals of both subunits of RuBisCO purified from C. vinosum were not identical to those deduced from the nt sequences, although they were completely the same as aa sequences deduced from rbcA-rbcB, another locus encoding RuBisCO in C. vinosum. Therefore, the rbcL-rbcS locus seems to be barely expressed under a standard condition for photoautotrophic growth. The homology of the nt sequences between rbcL and rbcA was 82%, and that between rbcS and rbcB was 63%, whereas the codon usages of these genes were basically identical. The rbcL-rbcS and rbcA-rbcB loci therefore must have evolved from a common ancestral set of genes after duplication, instead of lateral gene transfer.

• Valentin K, Zetsche K
• Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta.
• Plant Mol Biol. 1990; 15: 575-84
• Display abstract

• The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas phi. Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.

• Stein JL, Haygood M, Felbeck H
• Nucleotide sequence and expression of a deep-sea ribulose-1,5-bisphosphate carboxylase gene cloned from a chemoautotrophic bacterial endosymbiont.
• Proc Natl Acad Sci U S A. 1990; 87: 8850-4
• Display abstract

• The gene coding for ribulose-1,5-bisphosphate carboxylase [RuBisCO; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] was cloned from a sulfur-oxidizing chemoautotrophic bacterium that resides as an endosymbiont within the gill tissues of Alvinoconcha hessleri, a gastropod inhabiting deep-sea hydrothermal vents. Nucleotide sequence analysis of the cloned fragment demonstrated that the genes encoding the large (RbcL) and small (RbcS) subunits of the symbiont RuBisCO were organized similarly to the RuBisCO operons of free-living photo- and chemoautotrophic prokaryotes. The symbiont rbcL gene shared the highest degree of nucleotide sequence identity with the cyanobacterium Anabaena (69%) while the rbcS nucleotide sequence shared 61% identity with that of the green alga Chlamydomonas reinhardtii. Comparison with a 153-nucleotide partial rbcL sequence from a symbiont of the bivalve Solemya reidi indicated that the two symbiont sequences shared 85% sequence identity at the nucleotide level and 93% at the amino acid level, suggesting a relatively recent common origin. Escherichia coli transformed with a plasmid carrying the RuBisCO operon of the gastropod symbiont in the proper orientation for transcription from the plasmid lac promoter expressed catalytically active RuBisCO. The presence of enzyme activity suggests the proper assembly of the subunits of this deep-sea RuBisCO into the holoenzyme.

• von der Osten CH, Barbas CF 3rd, Wong CH, Sinskey AJ
• Molecular cloning, nucleotide sequence and fine-structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose-1,6-biphosphate aldolase to class I and class II aldolases.
• Mol Microbiol. 1989; 3: 1625-37
• Display abstract

• The Corynebacterium glutamicum fda gene encoding fructose-1,6-biphosphate (FBP) aldolase has been isolated by complementation of an Escherichia coli mutant. The nucleotide sequence of a 3371 bp chromosomal fragment containing the C. glutamicum fda gene was determined. The N-terminal amino acid sequence of C. glutamicum FBP aldolase identified the correct initiation site for the fda gene, and a molecular weight of 37,092 was predicted for the fda polypeptide. S1 nuclease mapping identified the transcriptional start site, and Northern hybridization analysis indicated that the fda gene encodes a single 1.3 kb transcript. The primary structure of C. glutamicum FBP aldolase shows strong homology to class II FBP aldolases. Conservation of primary structure was observed between class I and class II aldolases, but several residues essential for catalytic activity in class I aldolases were absent from class II aldolases.

• Alefounder PR, Baldwin SA, Perham RN, Short NJ
• Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli.
• Biochem J. 1989; 257: 529-34
• Display abstract

• Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.

• Gibson JL, Tabita FR
• Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides.
• J Bacteriol. 1988; 170: 2153-8
• Display abstract

• Two fructose 1,6-bisphosphatase structural genes (fbpA and fbpB) have been identified within two unlinked gene clusters that were previously shown to contain the Rhodobacter sphaeroides sequences that code for form I and form II ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoribulokinase. The fbpA and fbpB genes were localized to a region immediately upstream from the corresponding prkA and prkB sequences and were found to be transcribed in the same direction as the phosphoribulokinase and ribulose 1,5-bisphosphate carboxylase-oxygenase genes based on inducible expression of fructose 1,6-bisphosphatase activity directed by the lac promoter. A recombinant plasmid was constructed that contained the tandem fbpA and prkA genes inserted downstream from the lac promoter in plasmid pUC18. Both gene products were expressed in Escherichia coli upon induction of transcription with isopropyl beta-D-thiogalactoside, demonstrating that the two genes can be cotranscribed. A Zymomonas mobilis glyceraldehyde 3-phosphate-dehydrogenase gene (gap) hybridized to a DNA sequence located approximately 1 kilobase upstream from the form II ribulose 1,5-bisphosphate carboxylase-oxygenase gene. Although no corresponding gap sequence was found within the form I gene cluster, an additional region of homology was detected immediately upstream from the sequences that encode the form I and form II ribulose 1,5-bisphosphate carboxylase-oxygenases.

• Andersen K, Caton J
• Sequence analysis of the Alcaligenes eutrophus chromosomally encoded ribulose bisphosphate carboxylase large and small subunit genes and their gene products.
• J Bacteriol. 1987; 169: 4547-58
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• The nucleotide sequence of the chromosomally encoded ribulose bisphosphate carboxylase/oxygenase (RuBPCase) large (rbcL) and small (rbcS) subunit genes of the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was determined. We found that the two coding regions are separated by a 47-base-pair intergenic region, and both genes are preceded by plausible ribosome-binding sites. Cotranscription of the rbcL and rbcS genes has been demonstrated previously. The rbcL and rbcS genes encode polypeptides of 487 and 135 amino acids, respectively. Both genes exhibited similar codon usage which was highly biased and different from that of other organisms. The N-terminal amino acid sequence of both subunit proteins was determined by Edman degradation. No processing of the rbcS protein was detected, while the rbcL protein underwent a posttranslational loss of formylmethionyl. The A. eutrophus rbcL and rbcS proteins exhibited 56.8 to 58.3% and 35.6 to 38.5% amino acid sequence homology, respectively, with the corresponding proteins from cyanobacteria, eucaryotic algae, and plants. The A. eutrophus and Rhodospirillum rubrum rbcL proteins were only about 32% homologous. The N- and C-terminal sequences of both the rbcL and the rbcS proteins were among the most divergent regions. Known or proposed active site residues in other rbcL proteins, including Lys, His, Arg, and Asp residues, were conserved in the A. eutrophus enzyme. The A. eutrophus rbcS protein, like those of cyanobacteria, lacks a 12-residue internal sequence that is found in plant RuBPCase. Comparison of hydropathy profiles and secondary structure predictions by the method described by Chou and Fasman (P. Y. Chou and G. D. Fasman, Adv. Enzymol. 47:45-148, 1978) revealed striking similarities between A. eutrophus RuBPCase and other hexadecameric enzymes. This suggests that folding of the polypeptide chains is similar. The observed sequence homologies were consistent with the notion that both the rbcL and rbcS genes of the chemoautotroph A. eutrophus and the thus far characterized rbc genes of photosynthetic organisms have a common origin. This suggests that both subunit genes have a very ancient origin. The role of quaternary structure as a determinant of the rate of accepted amino acid substitution was examined. It is proposed that the sequence of the dimeric R. rubrum RuBPCase may be less conserved because there are fewer structural constraints for this RuBPCase than there are for hexadecameric enzymes.

• Goldschmidt-Clermont M, Rahire M
• Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii.
• J Mol Biol. 1986; 191: 421-32
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• We have sequenced the two genes for the small subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) in Chlamydomonas reinhardtii and analyzed their expression. The two genes encode variant small subunits that differ by four amino acid residues. Both genes are expressed and each is transcribed into an RNA of distinct size. The accumulation of the two RNAs changes depending on the growth conditions, so the small subunit composition of Rubisco may be expected to differ in response to the environment. The C. reinhardtii small subunit sequence is homologous to those of vascular plants or cyanobacteria, but is longer at the amino terminus and in internal positions. The number and location of the intervening sequences in the genes from C. reinhardtii and from other plants differ. In several cases, internal length differences in the polypeptide coincide with the positions of introns in the coding sequence. Thus, changes in the exon structure of the genes during evolution may have been accompanied by substantial changes in the encoded protein. The translation and splicing signals in C. reinhardtii are similar to those of other eukaryotes, but the transcription signals are less conserved and the highly biased codon usage is very unusual.

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