An Accurate Representation of the Number of bZIP Transcription Factors in the Triticum aestivum (Wheat) Genome and the Regulation of Functional Genes during Salt Stress.

Climate change is dramatically increasing the overall area of saline soils around the world, which is increasing by approximately two million hectares each year. Soil salinity decreases crop yields and, thereby, makes farming less profitable, potentially causing increased poverty and hunger in many areas. A solution to this problem is increasing the salt tolerance of crop plants. Transcription factors (TFs) within crop plants represent a key to understanding salt tolerance, as these proteins play important roles in the regulation of functional genes linked to salt stress. The basic leucine zipper (bZIP) TF has a well-documented role in the regulation of salt tolerance. To better understand how bZIP TFs are linked to salt tolerance, we performed a genome-wide analysis in wheat using the Chinese spring wheat genome, which has been assembled by the International Wheat Genome Sequencing Consortium. We identified 89 additional bZIP gene sequences, which brings the total of bZIP gene sequences in wheat to 237. The majority of these 237 sequences included a single bZIP protein domain; however, different combinations of five other domains also exist. The bZIP proteins are divided into ten subfamily groups. Using an in silico analysis, we identified five bZIP genes (ABF2, ABF4, ABI5, EMBP1, and VIP1) that were involved in regulating salt stress. By scrutinizing the binding properties to the 2000 bp upstream region, we identified putative functional genes under the regulation of these TFs. Expression analyses of plant tissue that had been treated with or without 100 mM NaCl revealed variable patterns between the TFs and functional genes. For example, an increased expression of ABF4 was correlated with an increased expression of the corresponding functional genes in both root and shoot tissues, whereas VIP1 downregulation in root tissues strongly decreased the expression of two functional genes. Identifying strategies to sustain the expression of the functional genes described in this study could enhance wheat's salt tolerance.

Improved Salinity Tolerance-Associated Variables Observed in EMS Mutagenized Wheat Lines.

Salinity tolerance-associated phenotypes of 35 EMS mutagenized wheat lines originating from BARI Gom-25 were compared. Vegetative growth was measured using non-destructive image-based phenotyping. Five different NaCl concentrations (0 to 160 mM) were applied to plants 19 days after planting (DAP 19), and plants were imaged daily until DAP 38. Plant growth, water use, leaf Na+, K+ and Cl- content, and thousand kernel weight (TKW) were measured, and six lines were selected for further analysis. In saline conditions, leaf Na+, K+, and Cl- content variation on a dry weight basis within these six lines were ~9.3, 1.4, and 2.4-fold, respectively. Relative to BARI Gom-25, two (OA6, OA62) lines had greater K+ accumulation, three (OA6, OA10, OA62) had 50-75% lower Na+:K+ ratios, and OA62 had ~30% greater water-use index (WUI). OA23 had ~2.2-fold greater leaf Na+ and maintained TKW relative to BARI Gom-25. Two lines (OA25, OA52) had greater TKW than BARI Gom-25 when grown in 120 mM NaCl but similar Na+:K+, WUI, and biomass accumulation. OA6 had relatively high TKW, high leaf K+, and WUI, and low leaf Na+ and Cl-. Phenotypic variation revealed differing associations between the parameters measured in the lines. Future identification of the genetic basis of these differences, and crossing of lines with phenotypes of interest, is expected to enable the assessment of which combinations of parameters deliver the greatest improvement in salinity tolerance.

Ligand Binding Site Comparison - LiBiSCo - a web-based tool for analyzing interactions between proteins and ligands to explore amino acid specificity within active sites.

Interaction between protein and ligands are ubiquitous in a biological cell, and understanding these interactions at the atom level in protein-ligand complexes is crucial for structural bioinformatics and drug discovery. Here, we present a web-based protein-ligand interaction application named Ligand Binding Site Comparison (LiBiSCo) for comparing the amino acid residues interacting with atoms of a ligand molecule between different protein-ligand complexes available in the Protein Data Bank (PDB) database. The comparison is performed at the ligand atom level irrespectively of having binding site similarity or not between the protein structures of interest. The input used in LiBiSCo is one or several PDB IDs of protein-ligand complex(es) and the tool returns a list of identified interactions at ligand atom level including both bonded and non-bonded interactions. A sequence profile for the interaction for each ligand atoms is provided as a WebLogo. The LiBiSco is useful in understanding ligand binding specificity and structural promiscuity among families that are structurally unrelated. The LiBiSCo tool can be accessed through https://albiorix.bioenv.gu.se/LiBiSCo/HomePage.py.

Identification and fine mapping of qGR6.2, a novel locus controlling rice seed germination under salt stress.

BACKGROUND: Rice growth is frequently affected by salinity. When exposed to high salinity, rice seed germination and seedling establishment are significantly inhibited. With the promotion of direct-seeding in Asia, improving rice seed germination under salt stress is crucial for breeding. RESULTS: In this study, an indica landrace Wujiaozhan (WJZ) was identified with high germinability under salt stress. A BC1F2 population derived from the crossing WJZ/Nip (japonica, Nipponbare)//Nip, was used to quantitative trait loci (QTL) mapping for the seed germination rate (GR) and germination index (GI) under H2O and 300 mM NaCl conditions. A total of 13 QTLs were identified, i.e. ten QTLs under H2O conditions and nine QTLs under salt conditions. Six QTLs, qGR6.1, qGR8.1, qGR8.2, qGR10.1, qGR10.2 and qGI10.1 were simultaneously identified under two conditions. Under salt conditions, three QTLs, qGR6.2, qGR10.1 and qGR10.2 for GR were identified at different time points during seed germination, which shared the same chromosomal region with qGI6.2, qGI10.1 and qGI10.2 for GI respectively. The qGR6.2 accounted for more than 20% of phenotypic variation under salt stress, as the major effective QTL. Furthermore, qGR6.2 was verified via the BC2F2 population and narrowed to a 65.9-kb region with eleven candidate genes predicted. Based on the microarray database, five candidate genes were found with high transcript abundances at the seed germination stage, of which LOC_Os06g10650 and LOC_Os06g10710 were differentially expressed after seed imbibition. RT-qPCR results showed the expression of LOC_Os06g10650 was significantly up-regulated in two parents with higher levels in WJZ than Nip during seed germination under salt conditions. Taken together, it suggests that LOC_Os06g10650, encoding tyrosine phosphatase family protein, might be the causal candidate gene for qGR6.2. CONCLUSIONS: In this study, we identified 13 QTLs from a landrace WJZ that confer seed germination traits under H2O and salt conditions. A major salt-tolerance-specific QTL qGR6.2 was fine mapped to a 65.9-kb region. Our results provide information on the genetic basis of improving rice seed germination under salt stress by marker-assisted selection (MAS).

Endogenous indole-3-acetamide levels contribute to the crosstalk between auxin and abscisic acid, and trigger plant stress responses in Arabidopsis.

The evolutionary success of plants relies to a large extent on their extraordinary ability to adapt to changes in their environment. These adaptations require that plants balance their growth with their stress responses. Plant hormones are crucial mediators orchestrating the underlying adaptive processes. However, whether and how the growth-related hormone auxin and the stress-related hormones jasmonic acid, salicylic acid, and abscisic acid (ABA) are coordinated remains largely elusive. Here, we analyse the physiological role of AMIDASE 1 (AMI1) in Arabidopsis plant growth and its possible connection to plant adaptations to abiotic stresses. AMI1 contributes to cellular auxin homeostasis by catalysing the conversion of indole-acetamide into the major plant auxin indole-3-acetic acid. Functional impairment of AMI1 increases the plant's stress status rendering mutant plants more susceptible to abiotic stresses. Transcriptomic analysis of ami1 mutants disclosed the reprogramming of a considerable number of stress-related genes, including jasmonic acid and ABA biosynthesis genes. The ami1 mutants exhibit only moderately repressed growth but an enhanced ABA accumulation, which suggests a role for AMI1 in the crosstalk between auxin and ABA. Altogether, our results suggest that AMI1 is involved in coordinating the trade-off between plant growth and stress responses, balancing auxin and ABA homeostasis.

Development and characterization of an EMS-mutagenized wheat population and identification of salt-tolerant wheat lines.

BACKGROUND: Triticum aestivum (wheat) is one of the world's oldest crops and has been used for >8000 years as a food crop in North Africa, West Asia and Europe. Today, wheat is one of the most important sources of grain for humans, and is cultivated on greater areas of land than any other crop. As the human population increases and soil salinity becomes more prevalent, there is increased pressure on wheat breeders to develop salt-tolerant varieties in order to meet growing demands for yield and grain quality. Here we developed a mutant wheat population using the moderately salt-tolerant Bangladeshi variety BARI Gom-25, with the primary goal of further increasing salt tolerance. RESULTS: After titrating the optimal ethyl methanesulfonate (EMS) concentration, ca 30,000 seeds were treated with 1% EMS, and 1676 lines, all originating from single seeds, survived through the first four generations. Most mutagenized lines showed a similar phenotype to BARI Gom-25, although visual differences such as dwarfing, giant plants, early and late flowering and altered leaf morphology were seen in some lines. By developing an assay for salt tolerance, and by screening the mutagenized population, we identified 70 lines exhibiting increased salt tolerance. The selected lines typically showed a 70% germination rate on filter paper soaked in 200 mM NaCl, compared to 0-30% for BARI Gom-25. From two of the salt-tolerant OlsAro lines (OA42 and OA70), genomic DNA was sequenced to 15x times coverage. A comparative analysis against the BARI Gom-25 genomic sequence identified a total of 683,201 (OA42), and 768,954 (OA70) SNPs distributed throughout the three sub-genomes (A, B and D). The mutation frequency was determined to be approximately one per 20,000 bp. All the 70 selected salt-tolerant lines were tested for root growth in the laboratory, and under saline field conditions in Bangladesh. The results showed that all the lines selected for tolerance showed a better salt tolerance phenotype than both BARI Gom-25 and other local wheat varieties tested. CONCLUSION: The mutant wheat population developed here will be a valuable resource in the development of novel salt-tolerant varieties for the benefit of saline farming.

Vesicles Are Persistent Features of Different Plastids.

Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage.

Chloroplast vesicle transport.

Photosynthesis is a well-known process that has been intensively investigated, but less is known about the biogenesis of the thylakoid membrane that harbors the photosynthetic machinery. Thylakoid membranes are constituted by several components, the major ones being proteins and lipids. However, neither of these two are produced in the thylakoid membranes themselves but are targeted there by different mechanisms. The interior of the chloroplast, the stroma, is an aqueous compartment that prevents spontaneous transport of single lipids and/or membrane proteins due to their hydrophobicities. Thylakoid targeted proteins are encoded either in the nucleus or plastid, and thus some cross the envelope membrane before entering one of the identified thylakoid targeting pathways. However, the pathway for all thylakoid proteins is not known. Lipids are produced at the envelope membrane and have been proposed to reach the thylakoid membrane by different means: invaginations of the envelope membrane, direct contact sites between these membranes, or through vesicles. Vesicles have been observed in chloroplasts but not much is yet known about the mechanism or regulation of their formation. The question of whether proteins can also make use of vesicles as one mechanism of transport remains to be answered. Here we discuss the presence of vesicles in chloroplasts and their potential role in transporting lipids and proteins. We additionally discuss what is known about the proteins involved in the vesicle transport and the gaps in knowledge that remain to be filled.

Arabidopsis RabF1 (ARA6) Is Involved in Salt Stress and Dark-Induced Senescence (DIS).

Arabidopsis small GTPase RabF1 (ARA6) functions in endosomal vesicle transport and may play a crucial role in recycling and degradation of molecules, thus involved in stress responses. Here we have reported that complementary overexpression lines RabF1OE (overexpression), GTPase mutants RabF1Q93L (constitutively active) and RabF1S47N (dominant negative) lines show longer root growth than wild-type, rabF1 knockout and N-myristoylation deletion (Δ1-29, N-terminus) complementary overexpression mutant plants under salt induced stress, which indicates that N-myristoylation of RabF1 is indispensable for salt tolerance. Moreover, RabF1 is highly expressed during senescence and RabF1OE lines were more tolerant of dark-induced senescence (DIS) than wild-type and rabF1.

The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth.

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high-phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO2 fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non-photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton-motive force across thylakoids. Small-angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long-range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild-type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro-organization of complexes and induction of photoprotective mechanisms.

Involvement of the electrophilic isothiocyanate sulforaphane in Arabidopsis local defense responses.

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue.

Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance.

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle: the signal recognition particle RNA is conserved in plastids of a wide range of photosynthetic organisms.

The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplast-specific protein cpSRP43. Nevertheless, homologs of the conserved SRP54 and the SRP receptor, FtsY, are present in higher plant chloroplasts. In this study, we analyzed the phylogenetic distribution of SRP components in photosynthetic organisms to elucidate the evolution of the SRP system. We identified conserved plastid SRP RNAs within all nonspermatophyte land plant lineages and in all chlorophyte branches. Furthermore, we show the simultaneous presence of cpSRP43 in these organisms. The function of this novel SRP system was biochemically and structurally characterized in the moss Physcomitrella patens. We show that P. patens chloroplast SRP (cpSRP) RNA binds cpSRP54 but has lost the ability to significantly stimulate the GTPase cycle of SRP54 and FtsY. Furthermore, the crystal structure at 1.8-Å resolution and the nucleotide specificity of P. patens cpFtsY was determined and compared with bacterial FtsY and higher plant chloroplast FtsY. Our data lead to the view that the P. patens cpSRP system occupies an intermediate position in the evolution from bacterial-type SRP to higher plant-type cpSRP system.

A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

Diversification of Plastid Structure and Function in Land Plants.

Plastids represent a largely diverse group of organelles in plant and algal cells that have several common features but also a broad spectrum of morphological, ultrastructural, biochemical, and physiological differences. Plastids and their structural and metabolic diversity significantly contribute to the functionality and developmental flexibility of the plant body throughout its lifetime. In addition to the multiple roles of given plastid types, this diversity is accomplished in some cases by interconversions between different plastids as a consequence of developmental and environmental signals that regulate plastid differentiation and specialization. In addition to basic plastid structural features, the most important plastid types, the newly characterized peculiar plastids, and future perspectives in plastid biology are also provided in this chapter.

Genome-Wide Analysis of MYB Transcription Factors in the Wheat Genome and Their Roles in Salt Stress Response.

Large and rapidly increasing areas of salt-affected soils are posing major challenges for the agricultural sector. Most fields used for the important food crop Triticum aestivum (wheat) are expected to be salt-affected within 50 years. To counter the associated problems, it is essential to understand the molecular mechanisms involved in salt stress responses and tolerance, thereby enabling their exploitation in the development of salt-tolerant varieties. The myeloblastosis (MYB) family of transcription factors are key regulators of responses to both biotic and abiotic stress, including salt stress. Thus, we used the Chinese spring wheat genome assembled by the International Wheat Genome Sequencing Consortium to identify putative MYB proteins (719 in total). Protein families (PFAM) analysis of the MYB sequences identified 28 combinations of 16 domains in the encoded proteins. The most common consisted of MYB_DNA-binding and MYB-DNA-bind_6 domains, and five highly conserved tryptophans were located in the aligned MYB protein sequence. Interestingly, we found and characterized a novel 5R-MYB group in the wheat genome. In silico studies showed that MYB transcription factors MYB3, MYB4, MYB13 and MYB59 are involved in salt stress responses. qPCR analysis confirmed upregulation of the expression of all these MYBs in both roots and shoots of the wheat variety BARI Gom-25 (except MYB4, which was downregulated in roots) under salt stress. Moreover, we identified nine target genes involved in salt stress that are regulated by the four MYB proteins, most of which have cellular locations and are involved in catalytic and binding activities associated with various cellular and metabolic processes.

Dimerization of TOC receptor GTPases and its implementation for the control of protein import into chloroplasts.

Pre-protein import into chloroplasts is facilitated by multiprotein translocon complexes in the envelope membranes. Major components of the TOC (translocon at the outer envelope membrane of chloroplasts) complex are the receptor proteins Toc33 and Toc159. These two receptors are related GTPases, and they are predicted to engage in homodimerization and/or heterodimerization. Although such dimerization has been studied extensively, its exact function in vivo remains elusive. In this issue of the Biochemical Journal, Oreb et al. present evidence that homodimerization of Toc33 prevents nucleotide exchange, thereby locking the receptor in the GDP-loaded state and preventing further activity. Pre-protein arrival is proposed to release this lock, through disruption of the dimer and subsequent nucleotide exchange. The Toc33-bound pre-protein is then able to progress to downstream steps in the translocation mechanism, with GTP hydrolysis defining another important control point as well as preparing the receptor for the next pre-protein client. These new results are discussed in the context of previous findings pertaining to TOC receptor dimerization and function.

Evolution and identification of DREB transcription factors in the wheat genome: modeling, docking and simulation of DREB proteins associated with salt stress.

Soil salinity and the resulting salt stress it imposes on crop plants is a major problem for modern agriculture. Understanding how salt tolerance mechanisms in plants are regulated is therefore important. One regulatory mechanism is the APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factor family, including dehydration responsive element binding (DREB) transcription factors. By binding to DNA, specifically upstream of genes that play roles in salt tolerance pathways, DREB proteins upregulate expression of these genes. DREB in Triticum aestivum (wheat) cluster in sub-groups and in this study by scanning the recently extended predicted proteome of wheat for DREB, we increased the number of members of this sub-family. Using the wheat genome, we identified 576 genes coding for the AP2 domain of which 508 were identified to have one AP2 domain, a characteristic of the DREB/ERF subfamily. We confirmed the existing four sub-groups by sequence-based phylogenetic analyses but also identified 32 new DREB subfamily members, not belonging to any known sub-group. Transcription factor profile inference analysis identified two genes, TraesCS2B02G002700 and TraesCS2D02G015200, being homologous to DREB1A of Arabidopsis thaliana. Based on molecular simulation (25 ns) analysis, TraesCS2B02G002700 with a CCGAC motif was observed to interact very stably with DNA. In silico mutational analysis at the 19th position in the DREB domain of TraesCS2B02G002700-DNA complex indicated this as a stable part for recognizing and forming interaction with DNA. Moreover, six target genes were predicted having an upstream CCGAC motif regulated by TraesCS2B02G002700. Our study provides an overall framework for exploring the transcription factors in plants and identifying e.g. potential salt stress target genes.Communicated by Ramaswamy H. Sarma.

MSALigMap-A Tool for Mapping Active-Site Amino Acids in PDB Structures onto Known and Novel Unannotated Homologous Sequences with Similar Function.

MSALigMap (Multiple Sequence Alignment Ligand Mapping) is a tool for mapping active-site amino-acid residues that bind selected ligands on to target protein sequences of interest. Users can also provide novel sequences (unavailable in public databases) for analysis. MSALigMap is written in Python. There are several tools and servers available for comparing and mapping active-site amino-acid residues among protein structures. However, there has not previously been a tool for mapping ligand binding amino-acid residues onto protein sequences of interest. Using MSALigMap, users can compare multiple protein sequences, such as those from different organisms or clinical strains, with sequences of proteins with crystal structures in PDB that are bound with the ligand/drug and DNA of interest. This allows users to easily map the binding residues and to predict the consequences of different mutations observed in the binding site. The MSALigMap server can be accessed at https://albiorix.bioenv.gu.se/MSALigMap/HomePage.py.

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane, is involved in thylakoid biogenesis.

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T-DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale-green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.