Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

Background

Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues.

Results

We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information.

Conclusion

For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments.

Reviewers

This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian.     

Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

Background

Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants.

Results

Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.

Conclusions

Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.     

Protein Functional Surfaces: Global Shape Matching and Local Spatial Alignments of Ligand Binding Sites

Background

Protein surfaces comprise only a fraction of the total residues but are the most conserved functional features of proteins. Surfaces performing identical functions are found in proteins absent of any sequence or fold similarity. While biochemical activity can be attributed to a few key residues, the broader surrounding environment plays an equally important role.

Results

We describe a methodology that attempts to optimize two components, global shape and local physicochemical texture, for evaluating the similarity between a pair of surfaces. Surface shape similarity is assessed using a three-dimensional object recognition algorithm and physicochemical texture similarity is assessed through a spatial alignment of conserved residues between the surfaces. The comparisons are used in tandem to efficiently search the Global Protein Surface Survey (GPSS), a library of annotated surfaces derived from structures in the PDB, for studying evolutionary relationships and uncovering novel similarities between proteins.

Conclusion

We provide an assessment of our method using library retrieval experiments for identifying functionally homologous surfaces binding different ligands, functionally diverse surfaces binding the same ligand, and binding surfaces of ubiquitous and conformationally flexible ligands. Results using surface similarity to predict function for proteins of unknown function are reported. Additionally, an automated analysis of the ATP binding surface landscape is presented to provide insight into the correlation between surface similarity and function for structures in the PDB and for the subset of protein kinases.     

QTL analysis in multiple sorghum populations facilitates the dissection of the genetic and physiological control of tillering

Key message

A QTL model for the genetic control of tillering in sorghum is proposed, presenting new opportunities for sorghum breeders to select germplasm with tillering characteristics appropriate for their target environments.

Abstract

Tillering in sorghum can be associated with either the carbon supply–demand (S/D) balance of the plant or an intrinsic propensity to tiller (PTT). Knowledge of the genetic control of tillering could assist breeders in selecting germplasm with tillering characteristics appropriate for their target environments. The aims of this study were to identify QTL for tillering and component traits associated with the S/D balance or PTT, to develop a framework model for the genetic control of tillering in sorghum. Four mapping populations were grown in a number of experiments in south east Queensland, Australia. The QTL analysis suggested that the contribution of traits associated with either the S/D balance or PTT to the genotypic differences in tillering differed among populations. Thirty-four tillering QTL were identified across the populations, of which 15 were novel to this study. Additionally, half of the tillering QTL co-located with QTL for component traits. A comparison of tillering QTL and candidate gene locations identified numerous coincident QTL and gene locations across populations, including the identification of common non-synonymous SNPs in the parental genotypes of two mapping populations in a sorghum homologue of MAX1, a gene involved in the control of tiller bud outgrowth through the production of strigolactones. Combined with a framework for crop physiological processes that underpin genotypic differences in tillering, the co-location of QTL for tillering and component traits and candidate genes allowed the development of a framework QTL model for the genetic control of tillering in sorghum.     

Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches

Background

BLAST is a commonly-used software package for comparing a query sequence to a database of known sequences; in this study, we focus on protein sequences. Position-specific-iterated BLAST (PSI-BLAST) iteratively searches a protein sequence database, using the matches in round i to construct a position-specific score matrix (PSSM) for searching the database in round i?+?1. Biegert and S?ding developed Context-sensitive BLAST (CS-BLAST), which combines information from searching the sequence database with information derived from a library of short protein profiles to achieve better homology detection than PSI-BLAST, which builds its PSSMs from scratch.

Results

We describe a new method, called domain enhanced lookup time accelerated BLAST (DELTA-BLAST), which searches a database of pre-constructed PSSMs before searching a protein-sequence database, to yield better homology detection. For its PSSMs, DELTA-BLAST employs a subset of NCBI??s Conserved Domain Database (CDD). On a test set derived from ASTRAL, with one round of searching, DELTA-BLAST achieves a ROC₅₀₀₀ of 0.270 vs. 0.116 for CS-BLAST. The performance advantage diminishes in iterated searches, but DELTA-BLAST continues to achieve better ROC scores than CS-BLAST.

Conclusions

DELTA-BLAST is a useful program for the detection of remote protein homologs. It is available under the ??Protein BLAST?? link at http://blast.ncbi.nlm.nih.gov.

Reviewers

This article was reviewed by Arcady Mushegian, Nick V. Grishin, and Frank Eisenhaber.     

Prediction of heme binding residues from protein sequences with integrative sequence profiles

Background

The heme-protein interactions are essential for various biological processes such as electron transfer, catalysis, signal transduction and the control of gene expression. The knowledge of heme binding residues can provide crucial clues to understand these activities and aid in functional annotation, however, insufficient work has been done on the research of heme binding residues from protein sequence information.

Methods

We propose a sequence-based approach for accurate prediction of heme binding residues by a novel integrative sequence profile coupling position specific scoring matrices with heme specific physicochemical properties. In order to select the informative physicochemical properties, we design an intuitive feature selection scheme by combining a greedy strategy with correlation analysis.

Results

Our integrative sequence profile approach for prediction of heme binding residues outperforms the conventional methods using amino acid and evolutionary information on the 5-fold cross validation and the independent tests.

Conclusions

The novel feature of an integrative sequence profile achieves good performance using a reduced set of feature vector elements.

     

Evidence of variability in the structure and recruitment of rhizospheric and endophytic bacterial communities associated with arable sweet sorghum (Sorghum bicolor (L) Moench)

Background and aims

Sorghum is the second most cultivated crop in Africa and is a staple food source in many African communities. Exploiting the associated plant growth-promoting bacteria (PGPB) has potential as an agricultural biotechnology strategy to enhance sorghum growth, yield and nutritional properties. Therefore this study aimed to evaluate factors that shape bacterial communities associated with sorghum farmed in South Africa, and to detect bacteria consistently associated with sorghum which may impart PGP activities.

Methods

Terminal-Restriction Fragment Length Polymorphism (T-RFLP) was used to assess factors that potentially shape rhizospheric (rhizosphere and rhizoplane) and endophytic (root, shoot, stem) bacterial communities associated with South African sorghum, and together with Denaturing Gradient Gel Electrophoresis (DGGE) to identify consistently sorghum-associated bacterial taxa.

Results

The sorghum rhizospheric communities were less variable than the endophytic ones. Geographical location was the main driver in describing bacterial community assemblages found in rhizospheric sorghum-linked niches, with total NO₃-N, NH₄-N, nitrogen, carbon, pH and, to a lesser extent, clay content identified as the main abiotic factors shaping sorghum-associated soil communities. Endophytic communities presented rather stochastic assemblages, with pH being the main variable explaining their structures. Despite community variations, specific bacterial taxa were consistently detected in sorghum-created rhizospheric and endophytic environments, irrespective of environmental factor effects.

Conclusions

Soil structure and composition, which are influenced by agricultural practices, played major roles in shaping sorghum-associated edaphic bacterial communities. In contrast, endophytic bacterial communities displayed more variation. Nevertheless, potentially agronomically relevant (cyano)bacterial taxa constantly associated with sorghum were identified which is suggestive of their deterministic recruitment.     

Genetic analysis of vegetative branching in sorghum

Key message

We identified quantitative trait loci influencing plant architecture that may be valuable in breeding of optimized genotypes for sustainable food and/or cellulosic biomass production, and advancing resilience to changing climates.

Abstract

We describe a 3-year study to identify quantitative trait loci (QTLs) for vegetative branching of sorghum in a recombinant inbred line population of 161 genotypes derived from two morphologically distinct parents, S. bicolor × S. propinquum. We quantify vegetative branching based on morphological position and physiological status. Different sets of QTLs for different levels of branching were identified. QTLs discovered on chromosomes 1, 3, 7 and 8 affect multiple vegetative branching variables, suggesting that these regions may contain genes that control general axillary meristem initiation. Other regions that only influence one vegetative branching trait could contain genes that influence developmental processes contributing to divergent patterns of plant architecture. We investigate the relationship between vegetative branching patterns and dry biomass, and conclude that tillers with mature panicles and immature secondary branches each show consistent positive correlation with dry biomass. Among 19 branching-related genes from rice, eight sorghum homologs of seven rice genes are in syntenic blocks within branching-related QTL likelihood intervals. Five of these eight genes are within 700 kb of SNPs significantly associated with differences in branching in genome-wide association study of a diversity panel of 377 sorghum accessions, and three contain striking allelic variations between S. bicolor and S. propinquum that are likely to impact gene functions. Unraveling genetic determinants for vegetative branching may contribute to deterministic breeding of optimized genotypes for sustainable food and cellulosic biomass production in both optimal and marginal conditions, which are resilient to future climates that are more volatile and more stressful.     

Phosphate aggravates iron chlorosis in sensitive plants grown on model calcium carbonate−iron oxide systems

Background and aims

The possible influence of phosphorus (P) on iron (Fe) deficiency chlorosis in susceptible plants needs elucidation. In this work, we tested the hypothesis that Fe chlorosis can be aggravated at high levels of P in the substrate.

Methods

Chickpea, lupin and peanut (in a preliminary experiment), and lupin and sorghum (in a second, factorial experiment) were successively grown on artificial substrates consisting of mixtures of Fe oxide-coated sand (FOCS), calcium carbonate (calcite) sand (CCS) and quartz sand to which phosphate was added at different doses.

Results

The proportion of FOCS in the substrate had a significant positive effect on leaf chlorophyll concentration (as estimated via SPAD) in all crops. In the factorial experiment, the SPAD value was negatively affected by the proportion of CCS in the dicot (lupin) but not in the monocot (sorghum). In the preliminary experiment, increasing the P dose generally had little effect on the SPAD of plants grown on the FOCS-rich substrate but a negative effect on those grown on the FOCS-poor substrate. In the factorial experiment, the P dose negatively affected SPAD in both lupin and sorghum.

Conclusions

Iron acquisition by the plant is negatively influenced by P probably because the solubility of the Fe oxides decreases with increasing coverage of their surfaces by sorbed phosphate.     

Biofortification of rice grain with zinc through zinc fertilization in different countries

Aims and background

The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Earlier, we reported that sorghum roots release higher BNI-activity when grown with NH ₄ ⁺ , but not with NO ₃ ^- as N source. Also for BNI release, rhizosphere pH of <5.0 is needed; beyond this, a negative effect on BNI release was observed with nearly 80% loss of BNI activity at pH >7.0. This study is aimed at understanding the inter-functional relationships associated with NH ₄ ⁺ uptake, rhizosphere-pH and plasma membrane H⁺-ATPase (PM H⁺-ATPase) activity in regulating the release of BNIs (biological nitrification inhibitors) from sorghum roots.

Methods

Sorghum was grown hydroponically and root exudates were collected from intact plants using a pH-stat system to separate the secondary acidification effects by NH ₄ ⁺ uptake on BNIs release. A recombinant luminescent Nitrosomonas europaea bioassay was used to determine BNI-activity. Root plasma membrane was isolated using a two-phase partitioning system. Hydrolytic H⁺-ATPase activity was determined. Split-root system setup was deployed to understand the localized responses to NH ₄ ⁺ , H⁺-ATPase-stimulator (fusicoccin) or H⁺-ATPase-inhibitor (vanadates) on BNI release by sorghum.

Results

Presence of NH ₄ ⁺ in the rhizosphere stimulated the expression of H⁺-ATPase activity and enhanced the release of BNIs from sorghum roots. Fusicoccin, which stimulates H⁺-ATPase activity, also stimulated BNIs release in the absence of NH ₄ ⁺ ; vanadate, which suppresses H⁺-ATPase activity, also suppressed the release of BNIs. NH ₄ ⁺ levels (in rhizosphere) positively influenced BNIs release and root H⁺-ATPase activity in the concentration range of 0-1.0 mM, indicating a close relationship between BNI release and root H⁺-ATPase activity with a possible involvement of carrier-mediated transport for the release of BNIs in sorghum.

Conclusion

Our results suggest that NH ₄ ⁺ uptake, PM H⁺-ATPase activity, and rhizosphere acidification are functionally inter-connected with BNI release in sorghum. Such knowledge is critical to gain insights into why BNI function is more effective in light-textured, mildly acidic soils compared to other soil types.     

Absence of carious lesions at margins of glass-ionomer cement and amalgam restorations: An update of systematic review evidence

Background

A recent analysis of protein sequences deposited in the NCBI RefSeq database indicates that ~8.5 million protein sequences are encoded in prokaryotic and eukaryotic genomes, where ~30% are explicitly annotated as "hypothetical" or "uncharacterized" protein. Our Comparison of Protein Active-Site Structures (CPASS v.2) database and software compares the sequence and structural characteristics of experimentally determined ligand binding sites to infer a functional relationship in the absence of global sequence or structure similarity. CPASS is an important component of our Functional Annotation Screening Technology by NMR (FAST-NMR) protocol and has been successfully applied to aid the annotation of a number of proteins of unknown function.

Findings

We report a major upgrade to our CPASS software and database that significantly improves its broad utility. CPASS v.2 is designed with a layered architecture to increase flexibility and portability that also enables job distribution over the Open Science Grid (OSG) to increase speed. Similarly, the CPASS interface was enhanced to provide more user flexibility in submitting a CPASS query. CPASS v.2 now allows for both automatic and manual definition of ligand-binding sites and permits pair-wise, one versus all, one versus list, or list versus list comparisons. Solvent accessible surface area, ligand root-mean square difference, and Cβ distances have been incorporated into the CPASS similarity function to improve the quality of the results. The CPASS database has also been updated.

Conclusions

CPASS v.2 is more than an order of magnitude faster than the original implementation, and allows for multiple simultaneous job submissions. Similarly, the CPASS database of ligand-defined binding sites has increased in size by ~ 38%, dramatically increasing the likelihood of a positive search result. The modification to the CPASS similarity function is effective in reducing CPASS similarity scores for false positives by ~30%, while leaving true positives unaffected. Importantly, receiver operating characteristics (ROC) curves demonstrate the high correlation between CPASS similarity scores and an accurate functional assignment. As indicated by distribution curves, scores ≥ 30% infer a functional similarity. Software URL: http://cpass.unl.edu.     

A split-root experiment shows that translocated phosphorus does not alleviate aluminium toxicity within plant tissue

Background and aims

My previous experimental findings suggested that phosphorus (P) enhances aluminium (Al) tolerance in both Al-tolerant and Al-sensitive wheat seedlings. However, the role of P in the amelioration of Al toxicity within plant tissue is still unclear. Therefore, a soil culture horizontal split-root system was used to quantify whether or not translocated P alleviates Al toxicity within the plant tissue.

Methods

Different level of Al and P were added in two compartments in various combinations for separate root halves. Constrasting Al-tolerant (ET8) and Al-sensitive (ES8) wheat genotypes were used as a testing plant.

Results

The limitation of root growth was independent to Al-toxicity in one root half. However, root proliferation occurred as a compensatory growth on the other root half that has no Al-toxicity. Where half of the roots were given 60 mg P/kg, plant did not translocated P in the other part of the root system that grown in Al toxic soil. When 40 mg P/kg were mixed with 60 mg AlCl₃/kg within one root half combinations, root dry weight of both ET8 and ES8 increased markedly in that root half. In contrast, root dry weight of both ET8 and ES8 decreased noticeably only 60 mg AlCl₃/kg treated root half. The shoot P and Al uptake in both ET8 and ES8 was lower in combined 40 mg P/kg and 60 mg AlCl₃/kg addition as compared to other combination with same P and Al level.

Conclusions

Result from this study confirm that addition of P to Al toxic acid soil played dual role like amelioration of Al-toxicity in soil and utilize P as nutrition for plant growth and development. Findings also attributed that added P was reduced by precipitation with added Al. However, evidence found that translocated P was not able to alleviate Al toxicity within plant tissue of both ES8 and ET8.     

Genome-wide patterns of genetic variation in sweet and grain sorghum (Sorghum bicolor)

Background

Sorghum (Sorghum bicolor) is globally produced as a source of food, feed, fiber and fuel. Grain and sweet sorghums differ in a number of important traits, including stem sugar and juice accumulation, plant height as well as grain and biomass production. The first whole genome sequence of a grain sorghum is available, but additional genome sequences are required to study genome-wide and intraspecific variation for dissecting the genetic basis of these important traits and for tailor-designed breeding of this important C₄ crop.

Results

We resequenced two sweet and one grain sorghum inbred lines, and identified a set of nearly 1,500 genes differentiating sweet and grain sorghum. These genes fall into ten major metabolic pathways involved in sugar and starch metabolisms, lignin and coumarin biosynthesis, nucleic acid metabolism, stress responses and DNA damage repair. In addition, we uncovered 1,057,018 SNPs, 99,948 indels of 1 to 10 bp in length and 16,487 presence/absence variations as well as 17,111 copy number variations. The majority of the large-effect SNPs, indels and presence/absence variations resided in the genes containing leucine rich repeats, PPR repeats and disease resistance R genes possessing diverse biological functions or under diversifying selection, but were absent in genes that are essential for life.

Conclusions

This is a first report of the identification of genome-wide patterns of genetic variation in sorghum. High-density SNP and indel markers reported here will be a valuable resource for future gene-phenotype studies and the molecular breeding of this important crop and related species.     

Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

Vesicle clustering by the molecular motor myosin XI enhances actin polymerization-dependent motility and polarized vesicle accumulation in tip-growing cells.     

Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

Background

Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology.

Methods

We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching.

Results

We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix.

Conclusions

The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.