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Muchero, Wellington

Publications and source records attributed to Muchero, Wellington.

31 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Bark morphological and chemical features are differentially correlated with disease resistance and yield in hybrid poplar taxa

In the southeastern United States, the establishment of short-rotation intensively cultured plantations of hybrid poplar has been hindered by its susceptibility to stem cankers. We evaluated the tradeoffs between biomass yield and disease tolerance in hybrid poplar genotypes belonging to P. deltoides × P. maximowiczii (DM), P. deltoides × P. nigra (DN), P. trichocarpa × P. maximowiczii (TM), and P. deltoides × P. deltoides (DD) taxa. We hypothesized that canker resistant genotypes will have thicker bark but bark thickness and biomass yield will be negatively correlated. After two growing seasons, the DD genotypes developed thicker bark compared to the genotypes of other taxa and bark thickness was not correlated with biomass yield in the DD genotypes (R 2 = 0.002). However, in the TM, DM, and DN genotypes, bark thickness was negatively correlated with biomass yield (R 2 = 0.33–0.77). Disease incidence studies revealed that the DM genotypes were most susceptible to canker whereas no disease was detected in DD genotypes. Furthermore, bark analysis conducted by Fourier transform infrared spectroscopy coupled with multivariate analysis showed that that DD genotypes to be chemically separate from the three hybrid genotypes and that bark chemistry was correlated with canker disease incidence. Taken together, these results reveal that it is possible to generate hybrid poplar genotypes with thicker bark, disease resistance, and higher biomass yields. This insight should guide further efforts to develop genetically improved hybrid poplar genotypes, both in terms of biomass yield and disease tolerance, for cultivation in the southeastern United States. Hybrid poplar cultivation in southeastern United States is hindered by its susceptibility to stem cankers. We evaluated tradeoffs between yield and canker disease resistance in various hybrid poplar genotypes. After two growing seasons, the DD genotypes showed disease resistance and developed thicker bark that was chemically distinct from the other genotypes. Bark thickness was not correlated with yield in the DD genotypes but was negatively correlated with yield in the other genotypes. These results will guide the development of hybrid poplar genotypes that are both disease resistant and high yielding for cultivation in the southeastern United States.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Plant myo -inositol transport influences bacterial colonization phenotypes

Plant microbiomes are assembled and modified through a complex milieu of biotic and abiotic factors. Despite dynamic and fluctuating contributing variables, specific host metabolites are consistently identified as important mediators of microbial interactions. We combine information from a large-scale metatranscriptomic dataset from natural poplar trees and experimental genetic manipulation assays in seedlings of the model plant Arabidopsis thaliana to converge on a conserved role for transport of the plant metabolite myo-inositol in mediating host-microbe interactions. While microbial catabolism of this compound has been linked to increased host colonization, we identify bacterial phenotypes that occur in both catabolism-dependent and -independent manners, suggesting that myo-inositol may additionally serve as a eukaryotic-derived signaling molecule to modulate microbial activities. Our data suggest host control of this compound and resulting microbial behavior are important mechanisms at play surrounding the host metabolite myo-inositol.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

GWAS identifies candidate genes controlling adventitious rooting in Populus trichocarpa

Adventitious rooting (AR) is critical to the propagation, breeding, and genetic engineering of trees. The capacity for plants to undergo this process is highly heritable and of a polygenic nature; however, the basis of its genetic variation is largely uncharacterized. To identify genetic regulators of AR, we performed a genome-wide association study (GWAS) using 1148 genotypes of Populus trichocarpa. GWASs are often limited by the abilities of researchers to collect precise phenotype data on a high-throughput scale; to help overcome this limitation, we developed a computer vision system to measure an array of traits related to adventitious root development in poplar, including temporal measures of lateral and basal root length and area. GWAS was performed using multiple methods and significance thresholds to handle non-normal phenotype statistics and to gain statistical power. These analyses yielded a total of 277 unique associations, suggesting that genes that control rooting include regulators of hormone signaling, cell division and structure, reactive oxygen species signaling, and other processes with known roles in root development. Numerous genes with uncharacterized functions and/or cryptic roles were also identified. These candidates provide targets for functional analysis, including physiological and epistatic analyses, to better characterize the complex polygenic regulation of AR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cell wall response of field grown Populus to Septoria infection

Due to its ability to spread quickly and result in tree mortality, Sphaerulina musiva (Septoria) is one of the most severe diseases impacting Populus . Previous studies have identified that Septoria infection induces differential expression of phenylpropanoid biosynthesis genes. However, more extensive characterization of changes to lignin in response to Septoria infection is lacking. To study the changes of lignin due to Septoria infection, four field grown, naturally variant Populus trichocarpa exhibiting visible signs of Septoria infection were sampled at health, infected, and reaction zone regions for cell wall characterization. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and acid hydrolysis were applied to identify changes to the cell wall, and especially lignin. FTIR and subsequent principal component analysis revealed that infected and reaction zone regions were similar and could be distinguished from the non-infected (healthy) region. NMR results indicated the general trend that infected region had a higher syringyl:guaiacyl ratio and lower p -hydroxybenzoate content than the healthy regions from the same genotype. Finally, Klason lignin content in the infected and/or reaction zone regions was shown to be higher than healthy region, which is consistent with previous observations of periderm development and metabolite profiling. These results provide insights on the response of Populus wood characteristics to Septoria infection, especially between healthy and infected region within the same genotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Split selectable marker systems utilizing inteins facilitate gene stacking in plants

The ability to stack multiple genes in plants is of great importance in the development of crops with desirable traits but can be challenging due to limited selectable marker options. Here we establish split selectable marker systems using protein splicing elements called “inteins” for Agrobacterium-mediated co-transformation in plants. First, we show that such a split selectable marker system can be used effectively in plants to reconstitute a visible marker, RUBY, from two non-functional fragments through tobacco leaf infiltration. Next, to determine the general applicability of our split selectable marker systems, we demonstrate the utility of these systems in the model plants Arabidopsis and poplar by successfully stacking two reporters eYGFPuv and RUBY, using split Kanamycin or Hygromycin resistance markers. In conclusion, this method enables robust plant co-transformation, providing a valuable tool for the simultaneous insertion of multiple genes into both herbaceous and woody plants efficiently.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis -elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CRISPR/Cas9-based gene activation and base editing in Populus

The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone “717-1B4” (P. tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g. those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone “717-1B4” and poplar clone “WV94” (P. deltoides “WV94”), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%–14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone “717-1B4”. Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Novel candidate genes for lignin structure identified through genome-wide association study of naturally varying Populus trichocarpa

Populus is a promising lignocellulosic feedstock for biofuels and bioproducts. However, the cell wall biopolymer lignin is a major barrier in conversion of biomass to biofuels. To investigate the variability and underlying genetic basis of the complex structure of lignin, a population of 409 three-year-old, naturally varying Populus trichocarpa genotypes were characterized by heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR). A subsequent genome-wide association study (GWAS) was conducted using approximately 8.3 million single nucleotide polymorphisms (SNPs), which identified 756 genes that were significantly associated (−log 10 ( p -value)>6) with at least one lignin phenotype. Several promising candidate genes were identified, many of which have not previously been reported to be associated with lignin or cell wall biosynthesis. These results provide a resource for gaining insights into the molecular mechanisms of lignin biosynthesis and new targets for future genetic improvement in poplar.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Regulating lignin biosynthesis and sugar release in plants

This disclosure provides genetically modified plants, plant cells and plant tissues that show modified lignin content and/or sugar release as compared to a wild type control plant which was not genetically modified. In addition, the disclosure provides methods of regulating lignin content and sugar release in a plant. The disclosure also provides methods of producing bioproducts using the genetically modified plants of the instant disclosure.

Xie, Meng↗
DOE OSTI2023

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone ‘52-225’ (P. trichocarpa × P. deltoides clone ‘52-225’). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

In planta single-molecule imaging and holographic force spectroscopy to study real-time, multimodal turnover dynamics of polysaccharides and associated carbohydrate metabolites

Cellular production of polysaccharides from simple sugar molecules serves critical roles in a variety of metabolic processes essential to the survival of every living organism. In the context of photosynthetically fixed carbon, cell wall polysaccharides synthesis is particularly of interest for increased biomass accumulation for human food, animal feed, and bioenergy related applications. Despite a long history of research in plant cell biology, our understanding of in planta cell wall biosynthesis and its regulation is far from complete due to lack of a comprehensive microscopy toolkit that encompass the multiple spatial and temporal scales in which cell wall polysaccharides fibrils are synthesized and assembled into intricate cell wall networks. In this project, a multidisciplinary team of scientists from Rutgers University, Vanderbilt University, and Oak Ridge National Laboratory carried out innovative multimodal, multiscale microscopy studies of cell wall synthesis using single-molecule force-spectroscopy, super-resolution fluorescence microscopy, and in vivo live cell imaging. Our novel multimodal and holistic imaging approach revealed plant cell wall polysaccharides synthesis processes in unprecedented detail across multiple spatiotemporal scales, from a single-molecule to a single-cell. Besides, the team has made technical and scientific innovations across multiple research fields—microscopy, bioengineering, single-molecule biophysics, and plant biology, etc.—to accomplish the goals. The results from this project will greatly advance the mechanistic and holistic understanding of in planta cell wall synthesis, which will accelerate the development of better transgenic crops for bioenergy applications. Moreover, the new toolbox, combining powerful advanced microscopy assays with cell/protein engineering, will have broader impacts on molecular and cellular biology fields by paving the way for studying cellular processes occurring across multiple physical scales with multimodal microscopy methodologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Lectin Receptor-like Kinase Signaling during Engineered Ectomycorrhiza Colonization

Mutualistic association can improve a plant’s health and productivity. G-type lectin receptor-like kinase (PtLecRLK1) is a susceptibility factor in Populus trichocarpa that permits root colonization by a beneficial fungus, Laccaria bicolor. Engineering PtLecRLK1 also permits L. bicolor root colonization in non-host plants similar to Populus trichocarpa. The intracellular signaling reprogramed by PtLecRLK1 upon recognition of L. bicolor to allow for the development and maintenance of symbiosis is yet to be determined. In this study, phosphoproteomics was utilized to identify phosphorylation-based relevant signaling pathways associated with PtLecRLK1 recognition of L. bicolor in transgenic switchgrass roots. Our finding shows that PtLecRLK1 in transgenic plants modifies the chitin-triggered plant defense and MAPK signaling along with a significant adjustment in phytohormone signaling, ROS balance, endocytosis, cytoskeleton movement, and proteasomal degradation in order to facilitate the establishment and maintenance of L. bicolor colonization. Moreover, protein–protein interaction data implicate a cGMP-dependent protein kinase as a potential substrate of PtLecRLK1.

ectomycorrhiza colonization↗