Plant Molecular Genetics Research Articles

BIOINFORMATIC ANALYSIS OF THE CODING SEQUENCES OF NICOTIANA SYLVESTRIS sHSP

During the evolution, plants have developed defense mechanisms against temperature stress associated with the synthesis of protective proteins, among which molecular chaperones predominate, in particular low molecular weight heat stress proteins (sHSPs). Despite the important role these proteins play in the defense response to high temperature stress, they are still poorly studied for many taxonomic groups of plants. In particular, almost nothing is known about the organization and diversity of heat shock genes/proteins in representatives of such an important genus as Nicotiana (tobacco). The article presents the results of a bioinformatic analysis of the multigene/multiprotein sHSP family in N. sylvestris, an important model object in plant physiology, biochemistry, molecular genetics and cell biotechnology. The GenBank database was used to search for homologous sequences using the BLAST algorithm. Amino acid sequences were aligned using the L-INS-I method on the MAFFT server. Phylogenetic analysis was performed by the maximum likelihood method using the PhyML plugin for Geneious Prime 2023.2.1. Statistical support of branches was calculated using the aLRT-Chi2 method. It was found that within the compared amino acid sequences of N. sylvestris sHSP, three parts can be distinguished: the variable N-terminal region, the conserved α-crystallin domain (ACD), and the C-terminal region. Based on the alignment of the sHSP amino acid sequences, a similarity dendrogram was constructed, on which several clades with high statistical support are visualized. The results of the comparative analysis of the amino acid sequences show that sHSPs of N. sylvestris belong to 10 structural classes. Proteins of seven classes are believed to be localized in the cytoplasm and/or nucleus, while the rest are located in the endoplasmic reticulum, mitochondria, plastids and peroxisomes. In total, according to the results of bioinformatic analysis, 24 genes encoding sHSP were found in the genome of N. sylvestris, as well as one pseudogene in which the fragment encoding the N-terminal region has been lost. Since the sequence of the pseudogene differs slightly from the closest representatives of this group, it can be assumed that the conversion of this region into a pseudogene is a relatively recent evolutionary event. Keywords: abiotic stress, bioinformatic analysis, genetic polymorphism, molecular genomics, molecular evolution, multigene family, α-crystallin domain, sHSP, N. sylvestris.

Assessment of genetic divergence in wheat lines (Triticum aestivum L.) involving biochemical and protein markers in rainfed conditions

Wheat (Triticum aestivum L.) is the important and strategic cereal crop for the majority of world’s populations. It is significant staple food of about two billion people. In next few years, world demand for wheat is expected to be 40 percent higher than that of its level today. Keeping in view the importance of the crop research work was conducted in the laboratory of Plant Breeding and Molecular Genetics, University of Poonch Rawalakot, AJK, Pakistan. The aim of the research was to find out the genetic diversity of different wheat lines. In the experiment, 50 different lines of wheat species (Triticum aestivum L.) was used to detect genetic diversity by utilizing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and biochemical analysis. On the basis of biochemical analysis lines 3111 and 3123 was diverse among 50 lines for antioxidant activity by using DPPH radical and 3135 and 3139 for phenolic contents and for flavonoid 3148 and 3107 was found more promising. Molecular characterization by SDS PAGE showed diversity in three wheat lines 3136, 3138 and 3110. These wheat lines could be our potential lines for future wheat improvement program as they were also promising regarding to the high yields.

Scalable, Flexible, and Cost-Effective Seedling Grafting.

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage seedlings of the small model plant, Arabidopsis thaliana, is technically challenging and time consuming due to the size and fragility of its seedlings. A growing collection of published methods describe this technique with varying success rates, difficulty, and associated costs. This paper describes a simple procedure to make an in-house reusable grafting device using silicone elastomer mix, and how to use this device for seedling grafting. At the time of this publication, each reusable grafting device costs only $0.47 in consumable materials to produce. Using this method, beginners can have their first successfully grafted seedlings in less than 3 weeks from start to finish. This highly accessible procedure will allow plant molecular genetics labs to establish seedling grafting as a normal part of their experimental process. Due to the full control users have in the creation and design of these grafting devices, this technique could be easily adjusted for use in larger plants, such as tomato or tobacco, if desired.

Research Progress and Application of Plant Branching

Plant branching development plays an important role in plant morphogenesis (aboveground plant type), the number and angle of branches are important agronomic characters that determine crop plant type. Effective branches determine the number of panicles or pods of crops and then control the yield of crops. With the rapid development of plant genomics and molecular genetics, great progress has been made in the study of branching development. In recent years, a series of important branching-related genes have been validated from Arabidopsis thaliana, rice, pea, tomato and maize mutants. It is reviewed that plant branching development is controlled by genetic elements and plant hormones, such as auxin, cytokinin and lactones (or lactone derivatives), as well as by environment and genetic elements. Meanwhile, shoot architecture in crop breeding was discussed in order to provide theoretical basis for the study of crop branching regulation.

Which field of research would Gregor Mendel choose in the 21st century?

Gregor Mendel's work on segregation of traits in plants established the basic methodology and rules of genetics. The interruption of Mendel's research activities in 1870 impeded the immediate recognition of the value of his work until the dawn of the 20th century. Only then were his founding laws of genetics validated, propelling the development of biological research toward the birth of molecular biology in the second half of the 20th century. While molecular plant genetics can be viewed as the spiritual heir of Mendel's research, one might wonder whether in the 21st century Gregor Mendel would prefer to practice scientific approaches other than molecular genetics such as population genetics, comparative genomics, or the emerging field of evo-chromo. In this perspective, I review aspects of these fields that might have attracted or perplexed a 21st century Mendel.

Broadening the horizon of crop research: a decade of advancements in plant molecular genetics to divulge phenotype governing genes.

Advancements in sequencing, genotyping, and computational technologies during the last decade (2011-2020) enabled new forward-genetic approaches, which subdue the impediments of precise gene mapping in varied crops. The modern crop improvement programs rely heavily on two major steps-trait-associated QTL/gene/marker's identification and molecular breeding. Thus, it is vital for basic and translational crop research to identify genomic regions that govern the phenotype of interest. Until the advent of next-generation sequencing, the forward-genetic techniques were laborious and time-consuming. Over the last 10 years, advancements in the area of genome assembly, genotyping, large-scale data analysis, and statistical algorithms have led faster identification of genomic variations regulating the complex agronomic traits and pathogen resistance. In this review, we describe the latest developments in genome sequencing and genotyping along with a comprehensive evaluation of the last 10-year headways in forward-genetic techniques that have shifted the focus of plant research from model plants to diverse crops. We have classified the available molecular genetic methods under bulk-segregant analysis-based (QTL-seq, GradedPool-Seq, QTG-Seq, Exome QTL-seq, and RapMap), target sequence enrichment-based (RenSeq, AgRenSeq, and TACCA), and mutation-based groups (MutMap, NIKS algorithm, MutRenSeq, MutChromSeq), alongside improvements in classical mapping and genome-wide association analyses. Newer methods for outcrossing, heterozygous, and polyploid plant genetics have also been discussed. The use of k-mers has enriched the nature of genetic variants which can be utilized to identify the phenotype-causing genes, independent of reference genomes. We envisage that the recent methods discussed herein will expand the repertoire of useful alleles and help in developing high-yielding and climate-resilient crops.

High-quality Arabidopsis thaliana Genome Assembly with Nanopore and HiFi Long Reads

Arabidopsis thaliana is an important and long-established model species for plant molecular biology, genetics, epigenetics, and genomics. However, the latest version of reference genome still contains a significant number of missing segments. Here, we reported a high-quality and almost complete Col-0 genome assembly with two gaps (named Col-XJTU) by combining the Oxford Nanopore Technologies ultra-long reads, Pacific Biosciences high-fidelity long reads, and Hi-C data. The total genome assembly size is 133,725,193 bp, introducing 14.6 Mb of novel sequences compared to the TAIR10.1 reference genome. All five chromosomes of the Col-XJTU assembly are highly accurate with consensus quality (QV) scores > 60 (ranging from 62 to 68), which are higher than those of the TAIR10.1 reference (ranging from 45 to 52). We completely resolved chromosome (Chr) 3 and Chr5 in a telomere-to-telomere manner. Chr4 was completely resolved except the nucleolar organizing regions, which comprise long repetitive DNA fragments. The Chr1 centromere (CEN1), reportedly around 9 Mb in length, is particularly challenging to assemble due to the presence of tens of thousands of CEN180 satellite repeats. Using the cutting-edge sequencing data and novel computational approaches, we assembled a 3.8-Mb-long CEN1 and a 3.5-Mb-long CEN2. We also investigated the structure and epigenetics of centromeres. Four clusters of CEN180 monomers were detected, and the centromere-specific histone H3-like protein (CENH3) exhibited a strong preference for CEN180 Cluster 3. Moreover, we observed hypomethylation patterns in CENH3-enriched regions. We believe that this high-quality genome assembly, Col-XJTU, would serve as a valuable reference to better understand the global pattern of centromeric polymorphisms, as well as the genetic and epigenetic features in plants.

New Honorary Member of the BSPP

Jonathan D. G. Jones grew up in London and graduated in Botany from Cambridge University followed by graduate work at the PBI, Cambridge. His career in plant–microbe interactions began with Ausubel at Harvard, studying Rhizobium–legume associations. He then worked at Advanced Genetic Sciences Inc. where his expertise using broad host-range plasmids underpinned the development of plant genetic engineering tools using Agrobacterium. Since 1988, he has led a research team at The Sainsbury Lab (TSL), Norwich, where he isolated the tomato Cf-9 gene for resistance to the fungus Cladosporium fulvum. Cf-9 encodes a pioneer cell surface “receptor-like protein” immune receptor, with extracellular leucine-rich repeats (LRRs). He isolated six such genes, each recognizing different fungal virulence factors. These preceded the Nobel prize-winning discoveries of similar Toll-like receptors in animal innate immunity. He went on to characterize complex immune receptor-encoding haplotypes with multiple paralogs and infer their evolutionary trajectories. He discovered that solvent-exposed amino acids in plant immune receptor LRRs are hypervariable and under diversifying selection for specific recognition of distinct pathogen molecules, and that unequal crossing over between paralogous genes at complex loci also contributes to receptor diversification. In a Cf-2 resistance gene suppressor screen, he discovered Rcr3, a cysteine protease required for Cf-2 to recognize the Avr2. In collaboration with the de Wit laboratory in Wageningen, he showed that Cf-2 “guards” Rcr3, activating defence when Rcr3 is inhibited by the Avr2 protease inhibitor. The resulting guard hypothesis was the subject of an influential paper with Dangl in 2001. He also identified several protein kinases and E3 ubiquitin ligases involved in Cf-dependent immunity, verifying their requirement for defence activation, and was first to show the role of receptor-like cytoplasmic kinases in cell surface receptor-mediated immunity. Most plant resistance genes encode intracellular nucleotide-binding LRR (NLR) immune receptors. He cloned one of the earliest (RPP5) and has defined >12 more. He was first to reveal the extreme haplotype diversity at the RPP5 locus between Arabidopsis accessions, again verifying extreme diversity in solvent-exposed amino acids of the LRRs of different paralogs, and unequal crossovers between paralogs in their evolution. In an influential 2006 paper with Dangl, he articulated the roles of cell surface “pattern-triggered immunity” and intracellular “effector-triggered immunity” in plant defence, integrating them for the first time. He has led discoveries on cross-talk between various hormone signalling pathways, in pathogen effector mechanisms, and in the role of variation in detection capacity in population resistance to disease. Some NLR receptors carry integrated domains (IDs) and are encoded adjacent in the genome to another NLR gene. Arabidopsis RPS4 and RRS1 are a paradigmatic example; RRS1 carries a WRKY transcription factor domain and the pair creates an immune receptor that detects pathogen effectors that act on WRKY domains. His work provided the first demonstration that NLRs can evolve to be immune receptors that contain IDs that mimic authentic pathogen targets. His fascination with genetic variation in host–pathogen interactions led him to develop sequence capture-based methods to interrogate plant immune receptor diversity. Resistance gene enrichment sequencing (RenSeq) has greatly accelerated our understanding of plant immune receptor repertoires. He also developed capture-based methods to investigate the genetic diversity of obligate parasites growing on hosts (PenSeq). He used RenSeq to address nonhost resistance (NHR) and defined the genetic basis of Arabidopsis NHR to Brassica-infecting races of white rust, revealing the crucial role of NLR receptors. Solanum americanum shows NHR to the potato late blight pathogen Phytophthora infestans. By isolating two NLR immune receptors, Rpi-amr1 and Rpi-amr3, from S. americanum, defining their recognized effectors from P. infestans, and verifying the efficacy of Rpi-amr1, Rpi-amr3, and Rpi-vnt1 in transgenic potato field experiments, he showed that their deployment might enable NHR to control blight in the field. He has played a key role in making the case that this approach to controlling crop disease is useful, safe, and sustainable. His discoveries in plant molecular genetics earned him election as Professor at UEA (1996), EMBO member (1998), Royal Society Fellow (2003), and International Member of US National Academy of Sciences (2015). He served in Royal Society working groups on food security and coauthored the Royal Society’s 2009 “Reaping the Benefits” report.

О.О. Созінов — видатний український агробіолог та організатор вітчизняної сільськогосподарської дослідної справи

Goal. To enlight the life and creative path of a prominent scientist in the field of plant genetics and selection academician O.O. Sozinov, to summarize his creative initiatives, achievements in the development of agricultural research in Ukraine. Methods. General scientific — analysis, synthesis, classification; historical — problem-chronological, comparatively historical, retrospective, biographical. Results. The contribution of the scientist to the formation of leading sectoral scientific institutions and organizations — the Selection and Genetic Institute — NSC of Seed Science and Variety Research of NAAS, Institute of General Genetics named after A.G. E. Vavilov, VASGNIL, NAAS, and others. The role is shown of O. Sozonov’s scientific school in the development of the basics of plant biochemical and molecular genetics. Conclusions. The scientific heritage of the scientist has been systematized in the following fields: genetics and selection, biology and morphology, anatomy and cytology of plants, agro phytocoenoses, agrobiotechnology, ecological problems of agriculture, seed production, problems of genomics.

Location, Location, Location: Phototropin 2 Action at the Chloroplast Membrane.

As someone with a background in plant molecular genetics, I confess to occasionally forgetting about location. Reverse genetics is a powerful tool for picking apart gene networks, but in which organ do these genes act? Which tissue of that organ? Which cells of that tissue and which compartment of

A Bridge to the World.

Zhi-Hong Xu is a plant physiologist who studied botany at Peking University (1959-1965). He joined the Shanghai Institute of Plant Physiology (SIPP), Chinese Academy of Sciences (CAS), as a graduate student in 1965. He recalls what has happened for the institute, during the Cultural Revolution, and he witnessed the spring of science eventually coming to China. Xu was a visiting scholar at the John Innes Institute and in the Department of Botany at Nottingham University in the United Kingdom (1979-1981). He became deputy director of SIPP in 1983 and director in 1991; he also chaired the State Key Laboratory of Plant Molecular Genetics SIPP (1988-1996). He worked as a visiting scientist in the Institute of Molecular and Cell Biology, National University of Singapore, for three months each year (1989-1992). He served as vice president of CAS (1992-2002) and as president of Peking University (1999-2008). Over these periods he was heavily involved in the design and implementation of major scientific projects in life sciences and agriculture in China. He is an academician of CAS and member of the Academy of Sciences for the Developing World. His scientific contributions mainly cover plant tissue culture, hormone mechanism in development, as well as plant developmental response to environment. Xu, as a scientist and leader who has made an impact in the community, called up a lot of excellent young scientists returning to China. His efforts have promoted the fast development of China's plant and agricultural sciences.

Genome Sequence of Fusarium oxysporum f. sp. matthiolae, a Brassicaceae Pathogen.

The filamentous fungus Fusarium oxysporum is a soilborne pathogen of many cultivated species and an opportunistic pathogen of humans. F. oxysporum f. sp. matthiolae is one of three formae speciales that are pathogenic to crucifers, including Arabidopsis thaliana, a premier model for plant molecular biology and genetics. Here, we report a genome assembly of F. oxysporum f. sp. matthiolae strain PHW726, generated using a combination of PacBio and Illumina sequencing technologies. The genome assembly presented here should facilitate in-depth investigation of F. oxysporum-Arabidopsis interactions and shed light on the genetics of fungal pathogenesis and plant immunity.

PIF4 Plays a Conserved Role in Solanum lycopersicum.

The adoption of Arabidopsis ( Arabidopsis thaliana ) as a model plant in the early 1980s led to a revolution in plant molecular genetics. Its diminutive size, rapid generation time, and small genome made Arabidopsis a fantastic tool, allowing us to build a complex picture of the genetic architecture

Replicating Arabidopsis Model Leaf Surfaces for Phyllosphere Microbiology

Artificial surfaces are commonly used in place of leaves in phyllosphere microbiology to study microbial behaviour on plant leaf surfaces. These surfaces enable a reductionist approach to be undertaken, to enable individual environmental factors influencing microorganisms to be studied. Commonly used artificial surfaces include nutrient agar, isolated leaf cuticles, and reconstituted leaf waxes. Recently, replica surfaces mimicking the complex topography of leaf surfaces for phyllosphere microbiology studies are appearing in literature. Replica leaf surfaces have been produced in agar, epoxy, polystyrene, and polydimethylsiloxane (PDMS). However, none of these protocols are suitable for replicating fragile leaves such as of the model plant Arabidopsis thaliana. This is of importance, as A. thaliana is a model system for molecular plant genetics, molecular plant biology, and microbial ecology. To overcome this limitation, we introduce a versatile replication protocol for replicating fragile leaf surfaces into PDMS. Here we demonstrate the capacity of our replication process using optical microscopy, atomic force microscopy (AFM), and contact angle measurements to compare living and PDMS replica A. thaliana leaf surfaces. To highlight the use of our replica leaf surfaces for phyllosphere microbiology, we visualise bacteria on the replica leaf surfaces in comparison to living leaf surfaces.

Tom Beeckman

Tom Beeckman

Induced Systemic Resistance (ISR) in Arabidopsis thaliana by Bacillus amyloliquefaciens and Trichoderma harzianum Used as Seed Treatments

The Trichoderma fungal species and the bacteria Bacillus species were described as inducers of plant systemic resistance in relation to their antagonistic activity. The objective of this study was to evaluate the effect of selected strains of Bacillus amyloliquefaciens (I3) and Trichoderma harzianum (A) on inducing systemic resistance in Arabidopsis thaliana as a model for plant molecular genetics. The microorganisms were identified and were confirmed for their antagonistic potential in vitro and in vivo in previous studies. In order to explore this mechanism, two mutants of A. thaliana carrying a PR1 promoter (a conventional marker of salicylic acid (SA) pathway) and LOX2 promoter (a marker triggering jasmonic acid (JA) pathway activation) were analyzed after inoculating antagonists. Transgenic reporter line analysis demonstrated that B. amyloliquefaciens I3 and T. harzianum A induce A. thaliana defense pathways by activating SA and JA at a high level compared to lines treated with chemical elicitors of references (acibenzolar-S-methyl (Bion 50 WG (water-dispersible granule)), SA, and methyl jasmonate). The efficacy of B. amyloliquefaciens I3 and T. harzianum A in inducing the defense mechanism in A. thaliana was demonstrated in this study.

A non-secreted plant defensin AtPDF2.6 conferred cadmium tolerance via its chelation in Arabidopsis.

Plant defensin AtPDF2.6 is not secreted to the apoplast and localized in cytoplasm. AtPDF2.6 is mainly expressed in root vascular bundles of xylem parenchyma cell, and significantly induced by Cd stress. AtPDF2.6 detoxicate cytoplasmic Cd via chelation, thus enhanced Cd tolerance in Arabidopsis. In order to detoxify the heavy metal cadmium (Cd), plants have evolved several mechanisms, among which chelation represents the major Cd-detoxification mechanism. In this study, we aimed to identify a new defensin protein involved in cytoplasmic Cd detoxification by using plant molecular genetics and physiological methods. The results of bioinformatic analysis showed that the Arabidopsis thaliana defensin gene AtPDF2.6 has a signal peptide that may mediate its secretion to the cell wall. Subcellular localization analysis revealed that AtPDF2.6 is localized to the cytoplasm and is not secreted to the apoplast, whereas histochemical analysis indicated that AtPDF2.6 is mainly expressed in the root xylem parenchyma cells and that its expression is significantly induced by Cd. An in vitro Cd-binding assay revealed that AtPDF2.6 has Cd-chelating activity. Heterologous overexpression of AtPDF2.6 increased Cd tolerance in Escherichia coli and yeast, and AtPDF2.6 overexpression significantly enhanced Cd tolerance in Arabidopsis, whereas functional disruption of AtPDF2.6 decreased Cd tolerance. These data suggest that AtPDF2.6 detoxifies cytoplasmic Cd via chelation and thereby enhances Cd tolerance in Arabidopsis. Our findings accordingly challenge the commonly accepted view of defensins as secreted proteins.

Linking genes with ecological strategies in Arabidopsis thaliana.

Arabidopsis thaliana is the most prominent model system in plant molecular biology and genetics. Although its ecology was initially neglected, collections of various genotypes revealed a complex population structure, with high levels of genetic diversity and substantial levels of phenotypic variation. This helped identify the genes and gene pathways mediating phenotypic change. Population genetics studies further demonstrated that this variation generally contributes to local adaptation. Here, we review evidence showing that traits affecting plant life history, growth rate, and stress reactions are not only locally adapted, they also often co-vary. Co-variation between these traits indicates that they evolve as trait syndromes, and reveals the ecological diversification that took place within A. thaliana. We argue that examining traits and the gene that control them within the context of global summary schemes that describe major ecological strategies will contribute to resolve important questions in both molecular biology and ecology.

A simple, inexpensive, scalable and low maintenance hydroponics system for growing halophyte: Lepidium sativum L. (Brassicaceae), ideal for manipulating salt stress and inferring gene expression levels

Soil salinity is a serious problem that limits productivity and survival of plants. Arabidopsis thaliana has served as a model plant to study different aspects of plant physiology and molecular genetics. However, its glycophytic nature, makes it unsuitable for salinity stress studies as it does not survive high dosage of salinity for extended periods. Moreover, the mechanisms of salt tolerance may be more evolved and possible different in halophytes. Hence, halophytes are better suited for such studies. With this background, the present investigation was initiated with the objectives of finding a suitable halophytic close relative of A. thaliana which may enable a better understanding of salt tolerance mechanisms. The present report describes the establishment of Lepidium sativum L. as a suitable halophyte for undertaking molecular physiology experiments under salt stress conditions. An efficient yet simple hydroponic culture system for L. sativum developed under the present investigation along with growth performance of the plant at varying levels of NaCl treatments (0–200 mM concentration) is also reported, showcasing its effective use in giving salt treatments that are amicable to any molecular biology laboratory involved in study of gene expression.

Phylogenetics and diversity analysis of Pennisetum species using Hemarthria EST‐SSR markers

AbstractExpressed sequence tag‐simple sequence repeat (EST‐SSR) markers are widely applied in plant molecular genetics studies due to their abundance in the genome, codominant nature, and high repeatability. To study the genetic diversity of 35 accessions and transferability of EST‐SSR markers in cross‐species applications, 21 primer pairs previously developed in Hemarthria were amplified across Pennisetum species. A total of 148 polymorphisms were generated with an average of 7.05 bands per locus. The mean values of the genetic parameters polymorphism information content, number of bands, effective number of bands, Nei's gene diversity, and Shannon's information index were 0.8430, 2.0000, 1.7640, 0.4247 and 0.6132, respectively. Cluster analysis of 35 accessions divided them into three clusters, which were consistent with dendrograms of 14 species. The among‐population component explained most of the genetic diversity (66%); the remaining variation (34%) occurred within populations. This study will provide more available EST‐SSR markers for Pennisetum species, and facilitate studies on germplasm collection and utilization of Pennisetum species.