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At least 127 records · Page 7

Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

Non-NASA Center↗

Isolation of potential plant growth-promoting bacteria from nodules of legumes grown in arid Botswana soil

As the world population increases, improvements in crop growth and yield will be needed to meet rising food demands, especially in countries that have not developed agricultural practices optimized for their own soils and crops. In many African countries, farmers improve agricultural productivity by applying synthetic fertilizers and pesticides to crops, but their continued use over the years has had serious environmental consequences including air and water pollution as well as loss of soil fertility. To reduce the overuse of synthetic amendments, we are developing inocula for crops that are based on indigenous soil microbes, especially those that enhance plant growth and improve agricultural productivity in a sustainable manner. We first isolated environmental DNA from soil samples collected from an agricultural region to study the composition of the soil microbiomes and then used Vigna unguiculata (cowpea), an important legume crop in Botswana and other legumes as “trap” plants using the collected soil to induce nitrogen-fixing nodule formation. We have identified drought-tolerant bacteria from Botswana soils that stimulate plant growth; many are species of Bacillus and Paenibacillus . In contrast, the cowpea nodule microbiomes from plants grown in these soils house mainly rhizobia particularly Bradyrhizobium , but also Methylobacterium spp. Hence, the nodule microbiome is much more limited in non-rhizobial diversity compared to the soil microbiome, but also contains a number of potential pathogenic bacteria.

Kosty, Melissa↗

Validation and functional characterization of transcription factors in wheat using cell-free protein expression and high-throughput sequencing technologies

Transcription factors (TFs) are critical biomolecules that control and regulate gene expression in every organism. In wheat, there are 3,606 genes annotated as putative TFs which are primarily uncharacterized. In this work, using a DAP-seq approach, we have validated and characterized a select number of TFs (5bl and 5dl) belonging to GeBP family of proteins, responsible for plant cellular growth, development, and differentiation. We found that top DNA binding motifs for 5bl and 5dl are (G/T)N(T/G)GTGGT and (C/G)AA(C/G)AA respectively. These motifs/peaks are enriched in the promoter regions of the genes, which are associated with rRNA biogenesis and protein maturation pathways. This study is also a first demonstration that DAP-seq can be applied for such a complex and large genome such as wheat.

59 BASIC BIOLOGICAL SCIENCES↗

Genomics-enabled analysis of specialized metabolism in bioenergy crops: Current progress and challenges

Plants produce a staggering diversity of specialized small molecule metabolites that play vital roles in mediating environmental interactions and stress adaptation. This chemical diversity derives from dynamic biosynthetic pathway networks that are often species-specific and operate under tight spatiotemporal and environmental control. A growing divide between demand and environmental challenges in food and bioenergy crop production have intensified research on these complex metabolite networks and their contribution to crop fitness. High-throughput omics technologies provide access to ever-increasing data resources for investigating plant metabolism. However, the efficiency of using such system-wide data to decode the gene and enzyme functions controlling specialized metabolism has remained limited; due largely to the recalcitrance of many plants to genetic approaches and the lack of ‘user-friendly’ biochemical tools for studying the diverse enzyme classes involved in specialized metabolism. With emphasis on terpenoid metabolism in the bioenergy crop switchgrass as an example, this review aims to illustrate current advances and challenges in the application of DNA synthesis and synthetic biology tools for accelerating the functional discovery of genes, enzymes and pathways in plant specialized metabolism. These technologies have accelerated knowledge development on the biosynthesis and physiological roles of diverse metabolite networks across many ecologically and economically important plant species and can provide resources for application to precision breeding and natural product metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.

NASA Discipline Plant Biology↗

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

NASA Discipline Plant Biology↗

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Non-NASA Center↗

Ectopic Expression of OLEOSIN 1 and Inactivation of GBSS1 Have a Synergistic Effect on Oil Accumulation in Plant Leaves

During the transformation of wild-type (WT) Arabidopsis thaliana, a T-DNA containing OLEOSIN-GFP (OLE1-GFP) was inserted by happenstance within the GBSS1 gene, resulting in significant reduction in amylose and increase in leaf oil content in the transgenic line (OG). The synergistic effect on oil accumulation of combining gbss1 with the expression of OLE1-GFP was confirmed by transforming an independent gbss1 mutant (GABI_914G01) with OLE1-GFP. The resulting OLE1-GFP/gbss1 transgenic lines showed higher leaf oil content than the individual OLE1-GFP/WT or single gbss1 mutant lines. Further stacking of the lipogenic factors WRINKLED1, Diacylglycerol O-Acyltransferase (DGAT1), and Cys-OLEOSIN1 (an engineered sesame OLEOSIN1) in OG significantly elevated its oil content in mature leaves to 2.3% of dry weight, which is 15 times higher than that in WT Arabidopsis. Inducible expression of the same lipogenic factors was shown to be an effective strategy for triacylglycerol (TAG) accumulation without incurring growth, development, and yield penalties.

59 BASIC BIOLOGICAL SCIENCES↗

A Radiation Chemistry Code Based on the Greens Functions of the Diffusion Equation

Ionizing radiation produces several radiolytic species such as.OH, e-aq, and H. when interacting with biological matter. Following their creation, radiolytic species diffuse and chemically react with biological molecules such as DNA. Despite years of research, many questions on the DNA damage by ionizing radiation remains, notably on the indirect effect, i.e. the damage resulting from the reactions of the radiolytic species with DNA. To simulate DNA damage by ionizing radiation, we are developing a step-by-step radiation chemistry code that is based on the Green's functions of the diffusion equation (GFDE), which is able to follow the trajectories of all particles and their reactions with time. In the recent years, simulations based on the GFDE have been used extensively in biochemistry, notably to simulate biochemical networks in time and space and are often used as the "gold standard" to validate diffusion-reaction theories. The exact GFDE for partially diffusion-controlled reactions is difficult to use because of its complex form. Therefore, the radial Green's function, which is much simpler, is often used. Hence, much effort has been devoted to the sampling of the radial Green's functions, for which we have developed a sampling algorithm This algorithm only yields the inter-particle distance vector length after a time step; the sampling of the deviation angle of the inter-particle vector is not taken into consideration. In this work, we show that the radial distribution is predicted by the exact radial Green's function. We also use a technique developed by Clifford et al. to generate the inter-particle vector deviation angles, knowing the inter-particle vector length before and after a time step. The results are compared with those predicted by the exact GFDE and by the analytical angular functions for free diffusion. This first step in the creation of the radiation chemistry code should help the understanding of the contribution of the indirect effect in the formation of DNA damage and double-strand breaks.

Plante, Ianik↗

A Computationally Efficient Algorithm for Sampling the Rudd Differential Cross Section

Monte Carlo radiation transport codes such as RITRACKS or Geant4 are used to simulate the interaction of ions with matter. These codes rely on sampling algorithms to determine interactions and various physical properties of particles involved in the simulations. It is crucial to develop efficient sampling algorithms since Monte Carlo radiation transport simulations can be time consuming. This work presents an efficient sampling algorithm to determine the energy of secondary electrons following ion-water interactions. The applicability and intended use of the algorithm are discussed in detail, and it is shown that the new algorithm is up to 6X10 4 times faster than the method currently used in Geant4-DNA.

Floriane Poignant↗

Simulation of Radiation-Induced DNA Damage With the Code RITRACKS

INTRODUCTION DNA damage is one of the most physiologically important effects of ionizing radiation. Clustered DNA damage events, like double-strand breaks (DSBs), have the most notable biological consequences. DNA damage types depend on both the track structure of the radiation and the spatial organization of the DNA. High linear energy transfer (LET) charged nuclei, found in galactic cosmic rays (GCR), are known to produce large numbers of complex DNA damage events. The human genome is packaged into chromatin, which can take on locus-dependent and cell type-dependent spatial conformations that correspond to epigenetic states, such as more open, extended structures in transcriptionally active chromatin. These epigenetic differences can affect DNA break patterns in response to ionizing radiation, potentially creating distinct DNA repair and signaling outcomes across the genome in different cells. MATERIAL AND METHODS The code RITRACKS (Relativistic Ion Tracks), which simulates stochastic radiation track structures and radiation chemistry, was used to model damage on isolated and histone-bound DNA by various types of ions and photons. The changes made to the code to perform radiation-induced DNA damage, and simulation results on single nucleosomes are given in our recent paper. In this work, the DNA building capabilities of RITRACKS have been extended to simulate more complex DNA structures build on the coarse-grain simulation framework meso-WLCsim. This code can sample generic chromatin fiber conformation ensembles based on the geometry of nucleosomes and mechanical properties of DNA. Using RITRACKS, we simulated the fragment length distributions (FLD) of irradiated DNA structures built using the chromatin conformations of WLCsim and obtained results representative of those obtained with Radiation-Induced Correlated Cleavage with sequencing (RICC-Seq) experiments [6]. We have also performed Fe ion and photon irradiations of K562, IMR90, BJ and RPE-1 cells at NSRL to experimentally validate results. Sample processing and data analysis are in progress and any available preliminary results will be discussed. DISCUSSION The recent updates in the code RITRACKS allow the calculation of several quantities such as the DNA damage yield and the FLD. This approach can be used to model epigenetic state-specific chromatin structure parameters to leverage the epigenetic state data available for many human cell types to infer relative DNA damage sensitivity among genomic loci.

I Plante↗

Impact of Soil Salinity on the Cowpea Nodule-Microbiome and the Isolation of Halotolerant PGPR Strains to Promote Plant Growth under Salinity Stress

Cowpea is one of the major legumes cultivated in arid and semiarid regions of the world. Four soil-microbial samples (SS-1 through SS-4) collected from semiarid soils in Punjab, Pakistan were planted with cowpea (Vigna unguiculata) crops, which were grown under salinity stress to analyze bacterial composition in the rhizosphere and within nodules using cultivation-dependent and -independent methods. Two varieties, 603 and the salt-tolerant CB 46, were each inoculated with or without the four different native soil samples or grown in medium either N-deficient (-N) or supplemented with N (+N). Plants inoculated with soil samples SS-2 and SS-4 grew better than plants inoculated with SS-1- and SS-3 and grew comparably with the +N controls. Environmental DNA (eDNA) was isolated from SS-1 and SS-4, and, by 16S ribosomal RNA sequencing, the soil microbiomes consisted mainly of Actinobacteria, Firmicutes, Proteobacteria, and other nonproteobacterial genera. However, analysis of eDNA isolated from cowpea nodules established by the trap plants showed that the nodule microbiome consisted almost exclusively of proteobacterial sequences, particularly species of Bradyrhizobium. Bacteria were isolated from both soils and nodules, and 34 of the 51 isolates tested positive for plant-growth-promoting rhizobacteria traits in plate assays. Many could serve as future inocula for crops in arid soils. The discrepancy between the types of bacteria isolated by culturing bacteria isolated from surface-sterilized cowpea nodules (proteobacteria and nonproteobacteria) versus those detected by sequencing DNA isolated from the nodules (proteobacteria) from cowpea nodules (proteobacteria and nonproteobacteria) versus those detected in the nodule microbiome (proteobacteria) needs further study.

59 BASIC BIOLOGICAL SCIENCES↗

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors

Abstract TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families.

59 BASIC BIOLOGICAL SCIENCES↗

Comprehensive SNP Data for 1,323 GWAS Population in Populus trichocarpa and Combined Annotation Files for P. trichocarpa v3.0 and v3.1

The VCF dataset includes genetic variations found in 1,323 Populus trichocarpa genotypes, providing valuable information for scientists studying plant genetics. Researchers have generated this dataset using whole-genome DNA short-read sequencing on the Illumina Genome Analyzer, HiSeq 2000, and HiSeq 2500 platforms. This sequencing effort ensured a minimum expected sequencing depth of 15×. The dataset comprises more than 9.7 million single nucleotide polymorphisms (SNPs) and indel variants. The combined annotation files are derived from P. trichocarpa v3.0 and v3.1. We merged these files to create a comprehensive annotation file used for GWAS analysis. In total, 38,830 genes overlapped between the two versions. For overlapping genes, we defined the start as the smaller and the end as the larger among the two versions to increase the likelihood of locating candidate genetic loci. Additionally, we included 2,505 unique genes from v3.0 and 4,120 unique genes from v3.1, resulting in a total of 45,455 genes in the updated annotation file.

09 BIOMASS FUELS↗