Biotechnological Manipulation Research Articles

Selection of <i>Cucumis sativus</i> L. for resistance to fusarium wilt using filtrate of the culture fluid of the fungus <i>Fusarium oxysporum</i> Schlectend

Relevance Traditional breeding methods are based on crossing and selection of genotypes among hybrid offspring. In recent decades, along with traditional methods, more and more attention is paid to alternative methods of selection, based on biotechnological manipulations with plants. One of the most important methods of biotechnology is the method of cell selection, which is based on the replacement of the whole plant, as a unit of selection, on its cell. Applying biotechnology techniques from a single plant can produce millions of cells, which increases the chances of finding, eliminating the need for areas for the cultivation of tested plants. As well as accelerating the selection process due to the possibility to carry out the study in the offseason. Methods The studies used the linear material of C. sativus hybrids of All-Russian Scientific Research Institute of Vegetable Growing – Branch of the FSBSI Federal Scientific Vegetable Center and Agroholding "Poisk". Plants were cultivated in laboratory room conditions. As explants used hypocotyl 0.5-1 cm segments isolated from young plants. Results To obtain Cucumis sativus plants with increased resistance to Fusarium by cell selection method, it is recommended to alternate culturing of callus on a non – selective medium containing sucrose in a concentration of 30 g/l, agar – 7 g/l, 0.1 mg/l, NUC – 0.5 mg/l and the filter of the cultural fluid of the fungus in a concentration of 10% within 3 passages.

Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.

Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.

Modification of Cassava Root Starch Phosphorylation Enhances Starch Functional Properties.

Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.

Bioaccessibility of Marine Carotenoids.

The benefit of carotenoids to human health is undeniable and consequently, their use for this purpose is growing rapidly. Additionally, the nutraceutical properties of carotenoids have attracted attention of the food industry, especially in a new market area, the ‘cosmeceuticals.’ Marine organisms (microalgae, seaweeds, animals, etc.) are a rich source of carotenoids, with optimal properties for industrial production and biotechnological manipulation. Consequently, several papers have reviewed the analysis, characterization, extraction and determination methods, biological functions and industrial applications. But, now, the bioaccessibility and bioactivity of marine carotenoids has not been focused of any review, although important achievements have been published. The specific and diverse characteristic of the marine matrix determines the bioavailability of carotenoids, some of them unique in the nature. Considering the importance of the bioavailability not just from the health and nutritional point of view but also to the food and pharmaceutical industry, we consider that the present review responds to an actual demand.

Multidisciplinary approaches for studying rhizobium-legume symbioses.

The rhizobium-legume symbiosis is a major source of fixed nitrogen (ammonia) in the biosphere. The potential for this process to increase agricultural yield while reducing the reliance on nitrogen-based fertilizers has generated interest in understanding and manipulating this process. For decades, rhizobium research has benefited from the use of leading techniques from a very broad set of fields, including population genetics, molecular genetics, genomics, and systems biology. In this review, we summarize many of the research strategies that have been employed in the study of rhizobia and the unique knowledge gained from these diverse tools, with a focus on genome- and systems-level approaches. We then describe ongoing synthetic biology approaches aimed at improving existing symbioses or engineering completely new symbiotic interactions. The review concludes with our perspective of the future directions and challenges of the field, with an emphasis on how the application of a multidisciplinary approach and the development of new methods will be necessary to ensure successful biotechnological manipulation of the symbiosis.

Activation of polyketide synthase gene promoter in Cannabis sativa by heterologous transcription factors derived from Humulus lupulus

Cannabis sativa, an annual herbaceous plant, produce wide variety of secondary metabolites among which delta-9-tetrahydrocannabinol (THC) is the most important one. The dissection of biosynthetic pathway(s) of this compound and its regulation by transcription factors (TFs) is an important prerequisite for efficient biotechnological manipulation of its secondary metabolome. A polyketide synthase (PKS) of C. sativa catalyzes the first step of cannabinoid biosynthesis, leading to the biosynthesis of olivetolic acid. Cloning and analysis of PKS promoter based on online PLACE, PlantCARE, and Genomatix Matinspector professional databases, indicated that PKS promoter consisted of cis-elements such as TATA-box, CAAT-box, W-box, Myb-box, E-box, and P-box. Plant expression vector PKS::GUS was constructed in such a way that the ATG of the PKS gene was in the frame with the β-glucuronidase (GUS) coding region. Using a combinatorial transient GUS expression system in Nicotiana benthamania leaves, it was shown that heterologous TFs such as HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1 from Humulus lupulus significantly activated PKS promoter. Moreover, Tombusvirus p19 core protein, which is known for silencing suppressor functions, acted in our combinatorial transient expression system as an enhancer of PKS promoter activity along with hop TFs. Our analyses suggested the involvement of the hop derived TFs (HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1A) and p19 in the activation of PKS gene promoter, which could be used for the genetic manipulation of C. sativa to enhance the cannabinoid production.

On biotechnology and animal welfare.

Abstract In the course of human history, the primary use of animals for more than 10 000 years has been agricultural - food, fibre, locomotion and power. And for most of that history, the well-being of the animals being used was assured by necessity. If one did not practice good husbandry, the animals did not produce, and those who raised the animals were equally harmed. Proper treatment of animals therefore was presuppositional to civilization. Unfortunately, with the coming of the Industrial Revolution, humans abandoned the 'ancient contract' with animals that had lasted 10 000 years. We could now increase the productivity of animals by use of 'technological sanders' that allowed us to keep animals under conditions where their welfare suffered but they continued to produce. The advent of biotechnology showed that this same exploitative mindset would dominate agricultural biotechnology. The rise of animal experimentation similarly demonstrated cavalier human disregard for animal well-being. Rationalizations such as the Cartesian claim that animals were machines buttressed this disregard. As was the case with agriculture, biotechnological manipulation of animals again disregarded animal welfare. For these reasons, it is morally necessary to set strict sense standards for genetic manipulation. Furthermore, public support for animal research is inversely proportional to the animals' degree of suffering. Thus, ethics and self-interest converge in this case.

The Role of Scientific Excellence in the Changing Meaning of Maize in Mexico

AbstractMaking policy is giving a meaning to objects. This perspective on policy making gained importance in the 1990s with the emergence of discursive approaches. In this article, we use the concepts of Hajer's discourse coalition approach to shed light on the evolving meaning of maize in Mexican society. Specifically, we trace a parallel between the evolution of biotechnology policy and discourses on maize over a 25‐year period. The article argues that until recently, the protection against biotechnological manipulations enjoyed by maize has been bolstered by a discourse granting the plant a special status in the country's history. However, the emergence of a new discourse grounded on the practice of scientific excellence is now challenging the old perspectives, and also finding support among government officials. As a result, the Mexican policies granting maize special protection is changing, and this policy change will likely trigger changes in the symbolic meaning of maize.

How do fins and limbs develop and evolve?

How do fish fins come into being and how do fins evolve into terrestrial tetrapod limbs? To answer this scientific question is substantially to understand how the genes that determine these traits are subject to natural and sexual selection, and how they thereby involve the innovation mechanics and evolution mechanisms with respect to development. Increasing evidence in recent 10 years suggests that fins might be as single fin-fold in early jawless fish, and then get makeable changes on the evolution roads from jawless fish to jaw fish and tetrapods, in which sequence divergence, expression pattern change, cooperative interaction, and even deletion of some required genes for fin development might play critical roles. The origin of fish fins and tetrapod limbs has been debated for more than three centuries from early natural philosophers and anatomists to modern developmental geneticists. In the recent 10 years, extensive and valuable data have been revealed from a lot of studies on paleontology, comparative anatomy and developmental genetics, and these complementary results have made us to understand the origin and underlying mechanisms behind the formation of the fish fin and tetrapod limb skeletons. It has been certified that fish has evolved to tetrapods and the multiple-basal bones of ancestral fish fins have been reduced to evolve into a single bone of tetrapod limb with fossil records. Similarly, during early embryogenesis, fish fins and tetrapod limbs share similar development mechanism, even though significant differences in histological, developmental and structural patterns exist between them. Many functional genes, such as Hox , And , Gli3 , Hand2 , play key roles in this transition or transformation. Natural selection and sex selection have led to sexual dimorphism of fish fins and morphological differences of tetrapod limbs. Along with the rapid development of genomics, genome editing and biotechnological manipulation techniques, the genetic basis of sexual dimorphism has been explored. Some gene families and signal pathways, such as Tbx gene family, Hox gene family, Ptch1 and its regulation elements, have been identified to involve in the morphological evolution. Significantly, it has been demonstrated that the differential expression of some sex-biased genes and its affected genes which are related with growth and gender differentiation may ultimately lead to sexual dimorphism of the fins. In this article, we have reviewed origin, evolutionary history and development patterns of fins, and elaborated molecule mechanisms on fin-to-limb evolution, sexual dimorphism of fish fins and morphological evolution of limbs. It is expected that a new era is coming for studies on developmental mechanisms and genetic basis of fin origin and fin-to-limb evolution, and some novel sights will be further provided.

Ensemble Architecture for Prediction of Enzyme-ligand Binding Residues Using Evolutionary Information.

Enzyme interactions with ligands are crucial for various biochemical reactions governing life. Over many years attempts to identify these residues for biotechnological manipulations have been made using experimental and computational techniques. The computational approaches have gathered impetus with the accruing availability of sequence and structure information, broadly classified into template-based and de novo methods. One of the predominant de novo methods using sequence information involves application of biological properties for supervised machine learning. Here, we propose a support vector machines-based ensemble for prediction of protein-ligand interacting residues using one of the most important discriminative contributing properties in the interacting residue neighbourhood, i. e., evolutionary information in the form of position-specific- scoring matrix (PSSM). The study has been performed on a non-redundant dataset comprising of 9269 interacting and 91773 non-interacting residues for prediction model generation and further evaluation. Of the various PSSM-based models explored, the proposed method named ROBBY (pRediction Of Biologically relevant small molecule Binding residues on enzYmes) shows an accuracy of 84.0 %, Matthews Correlation Coefficient of 0.343 and F-measure of 39.0 % on 78 test enzymes. Further, scope of adding domain knowledge such as pocket information has also been investigated; results showed significant enhancement in method precision. Findings are hoped to boost the reliability of small-molecule ligand interaction prediction for enzyme applications and drug design.

Exogenous Glutathione Modulates Salinity Tolerance of Soybean [Glycine max (L.) Merrill] at Reproductive Stage

In this study, we evaluated salt stress responses of 11 soybean genotypes and explored the protective roles of exogenous glutathione (GSH) against salt toxicity by assessing various biochemical and yield-attributing parameters. Exposure of the soybean genotypes to salt stress at the reproductive (R1) stage significantly decreased their yields by down-regulating the yield-contributing parameters as compared with unstressed controls. The highest decrease was found in number of seeds plant−1, yield plant−1, and number of pods plant−1, whereas the lowest decrease was recorded in number of seeds pod−1. Exogenous GSH was found to be effective in improving salinity tolerance, and the highest positive effects of GSH were recorded in terms of yield plant−1, number of seeds plant−1 and number of pods plant−1, whereas its lowest effect was observed with respect to the 100-seed weight. Cluster analysis of the genotypes based on yield-contributing and yield data revealed different levels of salt tolerance. Notably, BINA-01 and -02, and BINA-04 were recognized as the highest and lowest salt-tolerant genotypes, respectively. Furthermore, exogenous GSH alleviated oxidative stress in the representative contrasting genotypes at the vegetative (V3) stage by decreasing salt-induced accumulation of malondialdehyde and hydrogen peroxide. Taken together, our findings revealed that exogenous GSH application can minimize oxidative stress and contribute to the improvement of yield-contributing parameters, leading to improved yield in soybean genotypes under salt stress. Further investigations on molecular aspects will enable us to gain an in-depth understanding of how exogenous GSH can improve salinity tolerance in soybean, particularly at reproductive stage, to discover relevant pathways for biotechnological manipulation.

Metabolite profiling of the undifferentiated cultured cells and differentiated leaf tissues of Centella asiatica

Centella asiatica, a perennial herb renowned for its wide range of nutraceuticals properties, is a valuable source of plant secondary metabolites with various pharmacological activities. Novel approaches to develop alternatives for the production and enrichment of these secondary metabolites are receiving much attention, for example the biotechnological manipulation of undifferentiated cells cultures as a potential source of natural products. This study investigated the differences of the metabolite profiles between undifferentiated cells and differentiated leaf tissues of C. asiatica in comparison to a commercially available herbal medicine supplement. The secondary metabolites were extracted using methanol and analysed on an UHPLC-QTOF-MS platform. Metabolites were further identified and characterised based on their MS-fragmentation patterns and through comparison with authentic standards where available. Results revealed the similarities and dissimilarities and showed inter- and intra- relationships amid the different samples. A total of 18 metabolites including a number of hydroxycinnamic acid derivatives, flavonoids as well as the four centelloids (pentacyclic triterpenoids) were annotated in different sample groups across the different extracts. The results obtained verified that the different biological systems of C. asiatica are chemo-diverse, possibly related to regional and processing differences, and in the case of cells, to the level of cellular differentiation.

Sequence-based discrimination of protein-RNA interacting residues using a probabilistic approach

Protein interactions with ribonucleic acids (RNA) are well-known to be crucial for a wide range of cellular processes such as transcriptional regulation, protein synthesis or translation, and post-translational modifications. Identification of the RNA-interacting residues can provide insights into these processes and aid in relevant biotechnological manipulations. Owing to their eventual potential in combating diseases and industrial production, several computational attempts have been made over years using sequence- and structure-based information. Recent comparative studies suggest that despite these developments, many problems are faced with respect to the usability, prerequisites, and accessibility of various tools, thereby calling for an alternative approach and perspective supplementation in the prediction scenario. With this motivation, in this paper, we propose the use of a simple-yet-efficient conditional probabilistic approach based on the application of local occurrence of amino acids in the interacting region in a non-numeric sequence feature space, for discriminating between RNA interacting and non-interacting residues. The proposed method has been meticulously tested for robustness using a cross-estimation method showing MCC of 0.341 and F− measure of 66.84%. Upon exploring large scale applications using benchmark datasets available to date, this approach showed an encouraging performance comparable with the state-of-art. The software is available at https://github.com/ABCgrp/DORAEMON.

Identification of target genes and processes involved in erucic acid accumulation during seed development in the biodiesel feedstock Pennycress (Thlaspi arvense L.)

We studied erucic acid accumulation in the biodiesel feedstock Pennycress (Thlaspi arvense L.) as a first step towards the development of a sustainable strategy for biofuel production in the EU territory. To that end, two inbred Pennycress lines of European origin, “NASC” and “French,” were cultivated in a controlled chamber and in experimental field plots, and their growth, seed production and seed oil characteristics analyzed. Differences in some agronomical traits like vernalization (winter-French versus spring-NASC), flowering time (delayed in the French line) and seed production (higher in the French line) were detected. Both lines showed a high amount (35–39%) of erucic acid (22:1Δ13) in their seed oil. Biochemical characterization of the Pennycress seed oil indicated that TAG was the major reservoir of 22:1Δ13. Incorporation of 22:1Δ13 to TAG occurred very early during seed maturation, concomitant with a decrease of desaturase activity. This change in the acyl fluxes towards elongation was controlled by different genes at different levels. TaFAE1 gene, encoding the fatty acid elongase, seemed to be controlled at the transcriptional level with high expression at the early stages of seed development. On the contrary, the TaFAD2 gene that encodes the Δ12 fatty acid desaturase or TaDGAT1 that catalyzes TAG biosynthesis were controlled post-transcriptionally. TaWRI1, the master regulator of seed-oil biosynthesis, showed also high expression at the early stages of seed development. Our data identified genes and processes that might improve the biotechnological manipulation of Pennycress seeds for high-quality biodiesel production.

Abstract 1341: PRINT: A protein bioconjugation method with exquisite N-terminal specificity

Abstract The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation. We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients. We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. Citation Format: Surojit Sur, Yuan Qiao, Anja C. Fries, Robert N. O’Meally, Robert N. Cole, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. PRINT: A protein bioconjugation method with exquisite N-terminal specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1341.

HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses

Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth.

Application of reproductive technologies to improve dairy cattle genomic selection

Genomic selection implies assessing the breeding value of an animal based on the DNA markers uniformly covering the whole genome. This review summarizes the information on the modern trends in dairy cattle genomic selection and the use of reproductive technologies allowing for the increase of breeding efficiency. The main trends in the advance of genomic selection are the improvement of the accuracy of breeding value estimations by pooling the reference populations; use of female genotyping in breeding programs; imputation of genotypes based on lower marker density SNP arrays; and prediction of animal genotypes from the genotyped relatives. Genomic selection can be even more profitable in combination with the state-of-the-art reproductive biotechnologies, such as semen sexing, multiple ovulation and embryo transfer, ovum pick-up followed by in vitro fertilization, embryo genotyping, and cloning of the best breeders. In dairy cattle genomic selection programs, biotechnological manipulations with gametes and embryos allow for the improvement of many factors determining breeding efficiency, namely, selection intensity, accurate breeding value assessment, and the generation interval. Successful embryo genotyping methods for numerous markers after biopsy at the morula or blastocyst stage are based on the whole-genome amplification of the embryo’s DNA. Eventually, these achievements will provide the grounds for new approaches to reducing the generation interval, selecting elite cows, reducing the inbreeding rate, etc.

Comparative analyses of squalene synthase (SQS) proteins in poplar and pine by using bioinformatics tools

Squalene synthase (SQS, EC 2.5.1.21) is a major enzyme in biosynthesis of isoprenoid (farnesyl pyrophosphate (FPP) squalene). In the present study, we have analyzed SQS enzymes of black cottonwood (Populus trichocarpa, hereafter Pt) and Masson’s pine (Pinus massoniana, hereafter Pm) using bioinformatics tools. PtSQS and PmSQS sequences were found to have very similar physicochemical properties with “squalene/phytoene synthase” domain structure (PF00494). PtSQS sequence was 47.3 kDa weight and 413 amino acids long with a pI value of 6.86, while PmSQS was 46.6 kDa weight and 409 amino acids long with a pI of 7.92. Alignment of SQS protein sequences in 15 plant species showed a highly similar conserved pattern and included 77DTVED81 and 213DYLED217 motifs, which are rich in aspartic acids, for FPP binding sites. In phylogenetic tree, monocots and polycot were clearly separated from dicots with high bootstrap value (99 %). A total of 10 interaction partners were predicted for PtSQS and PmSQS proteins. Nine of them were hypothetical proteins (related with phytosterol biosynthesis), while one was putative uncharacterized protein. Similar 3D structures and identical binding sites were predicted for pine and poplar. In docking, FPP-PtSQS was found to make 8 H bonds with Asp81, Asp217, Glu80, and Gln206 residues in poplar with highest affinity while FPP-PmSQS made 7 H bonds with Arg49, Arg74, Ser48, and Val47 residues in pine with highest affinity. The results of this study will provide valuable theoretical knowledge for future studies of identification and characterization of SQS genes and proteins in various tree species and will provide an insight for studies of biotechnological manipulation of sterol biosynthesis pathway to enhance the plant stress tolerance and productivity.

New observations on gametogenic development and reproductive experimental tools to support seed yield improvement in cowpea [Vigna unguiculata (L.) Walp

Key messageCowpea reproductive tools.Vigna unguiculata L. Walp. (cowpea) is recognized as a major legume food crop in Africa, but seed yields remain low in most varieties adapted to local conditions. The development of hybrid cowpea seed that could be saved after each generation, enabling significant yield increases, will require manipulation of reproductive development from a sexual to an asexual mode. To develop new technologies that could support the biotechnological manipulation of reproductive development in cowpea, we examined gametogenesis and seed formation in two transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit distinct morphological and phenological traits but share a common developmental sequence in terms of ovule formation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to floral bud size and reproductive organ development, determining that gametogenesis occurs more rapidly in the anther than in the ovule. We also show that the mode of megagametogenesis is of the Polygonum-type and not Oenothera-type, as previously reported. Finally, we developed a whole-mount immunolocalization protocol and applied it to detect meiotic proteins in the cowpea megaspore mother cell, opening opportunities for comparing the dynamics of protein localization during male and female meiosis, as well as other reproductive events in this emerging legume model system. Electronic supplementary materialThe online version of this article (doi:10.1007/s00497-015-0273-3) contains supplementary material, which is available to authorized users.

RNA applications for endophytic research.

Endophytic microorganisms, mainly bacteria and fungi, have intrinsic relationships with the host plants, involving complex chemical and genetic communication networks. The relationship among these organisms involves the development of regulatory mechanisms of gene expression that control their development and response to different interactions. Although RNA molecules are already being used in studies of microorganism diversity and taxonomy, for example, using comparisons of rRNA regions, they may also be useful tools in the exploration of gene regulation and modeling of other molecules, such as the analysis of microRNA and small interfering RNAs. Transcriptional profile analyses are capable of providing robust information on biosynthetic pathways, genes involved in the interaction and differential production of metabolites by endophytes, using RNA-seq approaches. In-depth studies of RNA types and their functions in endophytes may provide valuable information that can be used for biotechnological manipulation of microorganisms to produce metabolites, bioremediation, biological control of pathogens, and decrease plant diseases, among other economically important applications. Our study highlights the present state of knowledge of studies involving endophytes, RNA molecules, and future perspectives.