Plant Molecular Biology Research Research Articles

Exogenous Brassinolide Ameliorates the Adverse Effects of Gamma Radiation Stress and Increases the Survival Rate of Rice Seedlings by Modulating Antioxidant Metabolism.

Gamma irradiation-based mutant creation is one of the most important methods for rice plant mutagenesis breeding and molecular biology research. Although median lethal dose irradiation severely damages rice seedlings, applying brassinolide (BR) can increase the survival rate of irradiated seedlings. In this study, we investigated the effects of soaking seeds in solutions containing different BR concentrations (0.001, 0.01, 0.1, 1.0, and 5.0 μmol/L) and then spraying the resulting seedlings twice with 0.1 μmol/L BR. The combined BR treatments markedly decreased the superoxide anion (O2•-), hydrogen peroxide (H2O2), and malondialdehyde contents but increased the chlorophyll content. An appropriate BR treatment of gamma-irradiated samples substantially increased the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and ascorbate peroxidase as well as the proline, ascorbic acid, and glutathione contents in rice seedling shoots. The BR treatment also promoted the growth of seedlings derived from irradiated seeds and increased the shoot and root fresh and dry weights. Most notably, soaking seeds in 0.01 or 0.1 μmol/L BR solutions and then spraying seedlings twice with 0.1 μmol/L BR significantly increased the final seedling survival rate and decreased mutant loss. The study results suggest that exogenous BR treatments can protect rice seedlings from gamma irradiation stress by enhancing antioxidant metabolism.

Biological Roles of Lipids in Rice.

Lipids are organic nonpolar molecules with essential biological and economic importance. While the genetic pathways and regulatory networks of lipid biosynthesis and metabolism have been extensively studied and thoroughly reviewed in oil crops such as soybeans, less attention has been paid to the biological roles of lipids in rice, a staple food for the global population and a model species for plant molecular biology research, leaving a considerable knowledge gap in the biological roles of lipids. In this review, we endeavor to furnish a current overview of the advancements in understanding the genetic foundations and physiological functions of lipids, including triacylglycerol, fatty acids, and very-long-chain fatty acids. We aim to summarize the key genes in lipid biosynthesis, metabolism, and transcriptional regulation underpinning rice's developmental and growth processes, biotic stress responses, abiotic stress responses, fertility, seed longevity, and recent efforts in rice oil genetic improvement.

The effect of phytosulfokine alpha on haploid embryogenesis and gene expression of Brassica napus microspore cultures.

Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

Genetic transformation in conifers: current status and future prospects.

Genetic transformation has been a cornerstone in plant molecular biology research and molecular design breeding, facilitating innovative approaches for the genetic improvement of trees with long breeding cycles. Despite the profound ecological and economic significance of conifers in global forestry, the application of genetic transformation in this group has been fraught with challenges. Nevertheless, genetic transformation has achieved notable advances in certain conifer species, while these advances are confined to specific genotypes, they offer valuable insights for technological breakthroughs in other species. This review offers an in-depth examination of the progress achieved in the genetic transformation of conifers. This discussion encompasses various factors, including expression vector construction, gene-delivery methods, and regeneration systems. Additionally, the hurdles encountered in the pursuit of a universal model for conifer transformation are discussed, along with the proposal of potential strategies for future developments. This comprehensive overview seeks to stimulate further research and innovation in this crucial field of forest biotechnology.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

A near-complete assembly of an Arabidopsis thaliana genome

The genome sequence of Arabidopsis thaliana, a widely adopted model species, has greatly expedited plant molecular biology research. Over 20 years after the first release of the genome sequence (Arabidopsis Genome Initiative, 2000), there remains unresolved gap regions that are presumably composed of highly repetitive sequences, such as telomeres, centromeres, 5S rDNA clusters, and nucleolar organizer regions (NORs) containing 45S rDNA. It is difficult to assemble such repeats using comparatively short sequencing reads.

Molecular communication network and its applications in crop sciences

Plant molecular biology and bacterial behaviour research in the future could focus on using genetically engineered bacteria as a sensor, hormonal/disease detector, and target gene expression, as well as establishing a bioluminescence feedback communication system. Over the last two decades, understanding plant signal transduction pathways of plant hormones has become an active research field to understand plant behavior better. To accomplish signal transduction, plants use a variety of hormones for inter- and intra-communication, and biotic or abiotic stressors activate those. Signal transduction pathways refer to the use of various communication methods by effectors to elicit a response at the molecular level. Research methodologies such as inter-kingdom signaling have been introduced to study signal transduction and communication pathways, or what we can term plant molecular communication. However, stochastic qualities are inherent in most technologies used to monitor these biological processes. Molecular communication (MC) is a new research topic that uses the natural features of biological organisms to communicate and aims to manipulate their stochastic nature to achieve the desired results. MC is a multidisciplinary research field inspired by the use of molecules to store, spread, and receive information between biological organisms known as "Biological Nanomachines." It has been used to demonstrate how biological entities may be characterised, modelled, and engineered as communication devices in the same manner as traditional communication technologies are. We attempted to link MC and PLANT'S MC in this studyand we believe that reasonable combined efforts may be made to use the functional applications of MC for detecting and understanding molecular-level activities such as signaling transduction pathways in crops.

Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max ).

Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.

Progress in Research on the Mechanisms Underlying Chloroplast-Involved Heat Tolerance in Plants.

Global warming is a serious challenge plant production has to face. Heat stress not only affects plant growth and development but also reduces crop yield and quality. Studying the response mechanisms of plants to heat stress will help humans use these mechanisms to improve the heat tolerance of plants, thereby reducing the harm of global warming to plant production. Research on plant heat tolerance has gradually become a hotspot in plant molecular biology research in recent years. In view of the special role of chloroplasts in the response to heat stress in plants, this review is focusing on three perspectives related to chloroplasts and their function in the response of heat stress in plants: the role of chloroplasts in sensing high temperatures, the transmission of heat signals, and the improvement of heat tolerance in plants. We also present our views on the future direction of research on chloroplast related heat tolerance in plants.

An efficient and rapid Rhizobium rhizogenes root transformation protocol for Lemna minor

Duckweeds belong to the smallest aquatic flowering plant family, Lemnaceae, and have a rapid doubling time, making this group an excellent system to study reduced morphology and wide environmental adaptability at the molecular level. Despite the availability of genomic and transcriptomic data for duckweed member, Lemna minor, lack of an efficient genetic transformation system has limited its use in plant molecular biology research. The present study reports an efficient and rapid Rhizobium rhizogenes-mediated root transformation system for L. minor. Two different factorial experiments were designed to test the effect of explant type, age, culture media and inoculation methods on transformation efficiency. Leaf and root tip cut explants were inoculated with R. rhizogenes strain MSU 440 harboring pBIN-YFP vector using yellow fluorescent protein (YFP) as a reporter gene for identification of transgenic roots. In addition, two different culture media, full MS and 0.25X Hoagland, and four different infection methods, solid culture, centrifugation, liquid culture and sonication, were compared. After 8 weeks, about 17% of the root tip-cut explants infected via the solid culture method and maintained in 0.25X Hoagland medium had YFP-expressing roots. These transgenic L. minor roots were morphologically similar to normal roots and PCR analysis demonstrated that the YFP-expressing roots were positive for the integration-expected rol genes. The described optimized root transformation procedure is a valuable tool for pursuing high-throughput gene characterization studies in L. minor.

A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense.

Acquiring high quality RNA is the basis of plant molecular biology research, plant genetics and other physiological investigations. At present, a large number of nucleotide isolation methods have been exploited or modified, such as commercial kits, CTAB, SDS methods and so on. Due to the nature of different plants, extraction methods vary. Moreover, efficiency of certain approach cannot be guaranteed due to composition of different plants and extracting high quality RNA from plants rich in polysaccharides and polyphenols are often difficult. The physical and chemical properties of polysaccharides which are similar to nucleic acids and other secondary metabolites will be coprecipitated with RNA irreversibly. Therefore, how to remove polysaccharides and other secondary metabolites during RNA extraction is the primary challenge. Dendrobium huoshanense is an Orchidaceae perennial herb that is rich in polysaccharides and other secondary metabolites. By using D. huoshanense as the subject, we improved the method originated from CHAN and made it suitable for plants containing high amount of polysaccharides and polyphenols. The extracted total RNA was clear and non-dispersive, with good integrity and no obvious contamination with DNA and other impurities. And it was also evaluated by gel electrophoresis, nucleic acid quantitative detector and PCR assessment. Thus, as a simple approach, it is suitable and efficient in RNA isolation for plants rich in polysaccharides and polyphenols.

Structural Properties of the RNA Synthesized by Glutamate Dehydrogenase for the Degradation of Total RNA

Glutamate dehydrogenase (GDH)-synthesized RNA, a nongenetic code-based RNA is suitable for unraveling the structural constraints imposed on the regulation (transcription, translation, siRNA etc.) of metabolism by genetic code. GDH-synthesized RNAs have been induced in whole plants to knock out target mRNA populations thereby producing plant phenotypes that are allergen-free; enriched in fatty acids, essential amino acids, shikimic acid, resveratrol etc. Methods applied hereunder for investigating the structural properties of GDH-synthesized RNA included purification of GDH isoenzymes, synthesis of RNA by the isoenzymes, reverse transcription of the RNA to cDNA, sequencing of the cDNA, computation of the G+C-contents, profiling the stability through PCR amplification compared with genetic code-based DNA; and biochemical characterization of the RNAs synthesized by individual hexameric isoenzymes of GDH. Single product bands resulted from the PCR amplification of the cDNAs of GDH-synthesized RNA, whereas several bands resulted from the amplification of genetic code-based DNA. The cDNAs have wide G+C-contents (35% to 59%), whereas genetic code-based DNA has narrower G+C-contents (50% to 60%). The GDH β6 homo-hexameric isoenzyme synthesized the A+U-rich RNAs, whereas the a6, and α6 homo-hexameric isoenzymes synthesized the G+C-rich RNAs. Therefore, the RNA synthesized by GDH is different from genetic code-based RNAs. In vitro chemical reactions revealed that GDH-synthesized RNA degraded total RNA to lower molecular weight products. Therefore, GDH-synthesized RNA is RNA enzyme. Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts. The RNA enzyme activity of GDH-synthesized RNA is ubiquitous in cells; it is readily induced by treatment of plants with mineral nutrients etc. and may simplify experimental approaches in plant enzymology and molecular biology research projects.

Investigation on Tree Molecular Genome of Arabidopsis Thaliana for Internet of Things

Research and regulation of tree growth at molecular level is a new trend in the development of forest biotechnology. However, as the limitations of wood as a research material, it is difficult to operate and compare the growth cycle in the laboratory, which greatly hinders study of the molecular biology of forest trees. Arabidopsis thaliana is the most important model plant in botanical research and is an excellent material for plant molecular biology research. Recent studies have shown that herbaceous Arabidopsis can not only provide genetic resources for tree molecular breeding but also can be used as an auxiliary means of wood molecular biology research and even can be directly used as an experimental model for the study of the molecular mechanism of tree development. This paper mainly discusses the establishment process of the tree molecular gene system platform under the background of the Internet of Things and studies the molecular genome of Arabidopsis, combining the characteristics of the tree itself and using the diversity of the frequency of different bases in the gene fragment to cluster the tree molecular genome, which is a molecular biology research and provides an important way of thinking.

Genome Editing Using Crispr/Cas System: New Era Genetic Technology in Agriculture to Boost Crop Output

Genome engineering with the RNA-guided CRISPR-Cas9 system in animals and plants is revolutionizing biology. First techniques of genome editing like zinc finger nucleases and synthetic nucleases called TALENs were a starting point but turned out to be expensive, difficult to handle and timeconsuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies. Moreover, these existing technologies depending on proteins as address labels and customizing new proteins for any new change to introduce in the DNA is a cumbersome process. Of the current generation of genome editing technologies, CRISPR-Cas9 is easier to use and more efficient and can be easily targeted to almost any genomic location of choice by a short RNA guide and has been successfully applied in many organisms, including model and crop plants. Together the system has the ability to detect specific sequences of letters within the genetic code and to cut DNA at a specific point. Simultaneously with other sequence-specific nucleases, CRISPR/ Cas9 has already breach the boundaries and made genetic engineering much more versatile, efficient and easy. There really doesn’t seem to be a limit in applications of CRISPR system extendable from bacteria to complex eukaryotic organisms including plants changing the pace and course of agricultural, Biomedicine and Biotechnological research in the future. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology.

UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts.

BackgroundThe complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays.ResultsWe established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts.ConclusionsThe described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0142-6) contains supplementary material, which is available to authorized users.

Genome editing with engineered nucleases in plants.

Numerous examples of successful 'genome editing' now exist. Genome editing uses engineered nucleases as powerful tools to target specific DNA sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as genome editing tools, in the form of designed ZF nucleases (ZFNs). More recently, transcription activator-like effector nucleases (TALENs), as well as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes RNA-DNA interactions, have proved useful. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved by custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end-joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR) to occur efficiently at specific sites in the genome. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology.

From jellyfish to biosensors: the use of fluorescent proteins in plants

The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors.

Molecular Mechanism of Heavy Metal Toxicity and Tolerance in Plants: Central Role of Glutathione in Detoxification of Reactive Oxygen Species and Methylglyoxal and in Heavy Metal Chelation

Heavy metal (HM) toxicity is one of the major abiotic stresses leading to hazardous effects in plants. A common consequence of HM toxicity is the excessive accumulation of reactive oxygen species (ROS) and methylglyoxal (MG), both of which can cause peroxidation of lipids, oxidation of protein, inactivation of enzymes, DNA damage and/or interact with other vital constituents of plant cells. Higher plants have evolved a sophisticated antioxidant defense system and a glyoxalase system to scavenge ROS and MG. In addition, HMs that enter the cell may be sequestered by amino acids, organic acids, glutathione (GSH), or by specific metal-binding ligands. Being a central molecule of both the antioxidant defense system and the glyoxalase system, GSH is involved in both direct and indirect control of ROS and MG and their reaction products in plant cells, thus protecting the plant from HM-induced oxidative damage. Recent plant molecular studies have shown that GSH by itself and its metabolizing enzymes—notably glutathioneS-transferase, glutathione peroxidase, dehydroascorbate reductase, glutathione reductase, glyoxalase I and glyoxalase II—act additively and coordinately for efficient protection against ROS- and MG-induced damage in addition to detoxification, complexation, chelation and compartmentation of HMs. The aim of this review is to integrate a recent understanding of physiological and biochemical mechanisms of HM-induced plant stress response and tolerance based on the findings of current plant molecular biology research.

Ammonia increases nitric oxide, free Zn2+and metallothionein mRNA expression in cultured rat astrocytes

Objective Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences. Topics general biochemistry pathobiochemistry structural biology molecular and cellular biology genetics and epigenetics molecular medicine cancer research virology immunology plant molecular biology and biochemistry novel experimental methodologies Article formats Research Articles, Short Communications, Reviews and Minireviews Reviews are published by invitation only, but suggestions to the Editor-in-Chief are welcome. The journal is associated with the German Society for Biochemistry and Molecular Biology (GBM).

Highlight: The Winter School on Proteinases and Their Inhibitors

Objective Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences. Topics general biochemistry pathobiochemistry structural biology molecular and cellular biology genetics and epigenetics molecular medicine cancer research virology immunology plant molecular biology and biochemistry novel experimental methodologies Article formats Research Articles, Short Communications, Reviews and Minireviews Reviews are published by invitation only, but suggestions to the Editor-in-Chief are welcome. The journal is associated with the German Society for Biochemistry and Molecular Biology (GBM).