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Ding, Shi-You

Publications and source records attributed to Ding, Shi-You.

17 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. For this work, we found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5 G43R + S49T+S54P+S80A+Y88F . Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Adaptative Survival of Aspergillus fumigatus to Echinocandins Arises from Cell Wall Remodeling Beyond β-1,3-glucan Synthesis Inhibition

Antifungal echinocandins inhibit the biosynthesis of β-1,3-glucan, a major and essential polysaccharide component of the fungal cell wall. However, the efficacy of echinocandins against the pathogen Aspergillus fumigatus is limited. Here, we use solid-state nuclear magnetic resonance (ssNMR) and other techniques to show that echinocandins induce dynamic changes in the assembly of mobile and rigid polymers within the A. fumigatus cell wall. The reduction of β-1,3- glucan induced by echinocandins is accompanied by a concurrent increase in levels of chitin, chitosan and highly polymorphic α-1,3-glucans, whose physical association with chitin maintains cell wall integrity and modulates water permeability. The rearrangement of the macromolecular network is dynamic and controls the permeability and circulation of the drug throughout the cell wall. Thus, our results indicate that echinocandin treatment triggers compensatory rearrangements in the cell wall that may help A. fumigatus to tolerate the drugs’ antifungal effects.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Xylan Is Critical for Proper Bundling and Alignment of Cellulose Microfibrils in Plant Secondary Cell Walls

Plant biomass represents an abundant and increasingly important natural resource and it mainly consists of a number of cell types that have undergone extensive secondary cell wall (SCW) formation. These cell types are abundant in the stems of Arabidopsis, a well-studied model system for hardwood, the wood of eudicot plants. The main constituents of hardwood include cellulose, lignin, and xylan, the latter in the form of glucuronoxylan (GX). The binding of GX to cellulose in the eudicot SCW represents one of the best-understood molecular interactions within plant cell walls. The evenly spaced acetylation and 4-O-methyl glucuronic acid (MeGlcA) substitutions of the xylan polymer backbone facilitates binding in a linear two-fold screw conformation to the hydrophilic side of cellulose and signifies a high level of molecular specificity. However, the wider implications of GX–cellulose interactions for cellulose network formation and SCW architecture have remained less explored. In this study, we seek to expand our knowledge on this by characterizing the cellulose microfibril organization in three well-characterized GX mutants. The selected mutants display a range of GX deficiency from mild to severe, with findings indicating even the weakest mutant having significant perturbations of the cellulose network, as visualized by both scanning electron microscopy (SEM) and atomic force microscopy (AFM). We show by image analysis that microfibril width is increased by as much as three times in the severe mutants compared to the wild type and that the degree of directional dispersion of the fibrils is approximately doubled in all the three mutants. Further, we find that these changes correlate with both altered nanomechanical properties of the SCW, as observed by AFM, and with increases in enzymatic hydrolysis. Results from this study indicate the critical role that normal GX composition has on cellulose bundle formation and cellulose organization as a whole within the SCWs.

09 BIOMASS FUELS↗
DOE OSTI2021

Visualizing plant cell wall changes proves the superiority of hydrochloric acid over sulfuric acid catalyzed γ-valerolactone pretreatment

Thermochemical pretreatment is one of the key steps to process lignocellulosic biomass for production of biofuels and biomaterials. γ-valerolactone (GVL) has been used as a green and renewable solvent to efficiently dissolve lignin under acidic conditions and enhance subsequent enzyme digestibility. Further improvement is still required to lower the pretreatment temperature in order to reduce sugar degradation and irreversible lignin condensation, as well as the capital cost. In this study, we compared the use of HCl and H 2 SO 4 as catalysts during GVL pretreatment, and found the performance of GVL-HCl at 100 °C was comparable to that of GVL-H 2 SO 4 at 120 °C in terms of xylan and lignin removal and enzyme digestibility. We further monitored the lignin removal and cellulose accessibility in the cell walls at different pretreatment time points by imaging the changes in lignin auto-fluorescence and in CtCBM3-GFP binding with confocal laser scanning microscopy (CLSM), respectively. We found GVL-HCl pretreatment at a relative low temperature (100 °C) could rapidly remove lignin in the compound middle lamella and cell corner areas, which concurred with the increase of CtCBM3-GFP binding in these areas. Real-time imaging of cell wall degradation by cellulases further revealed that the secondary cell walls could be digested from both cell lumen and CML sides, and eventually fully deconstructed within 24 h. Furthermore, our results provide new insight into the effects of chloride anions on plant cell wall structure during GVL pretreatment, and offer potential routes to further optimize and enhance the efficiency of GVL-based pretreatment.

09 BIOMASS FUELS↗
DOE OSTI2019

Imaging Changes in Cell Walls of Engineered Poplar by Stimulated Raman Scattering and Atomic Force Microscopy

Lignin is a major impediment in the deconstruction of plant biomass to its soluble monomeric constituents. Modification of the lignin biosynthetic pathway has proven to be an effective means of reducing biomass recalcitrance but can often result in impaired growth. As one of the successful instances of “designer” biomass, “Zip-lignin” has been generated in poplar to introduce ester linkages into the lignin backbone. The resulting plants grow normally and have improved biomass deconstructability, especially under mild alkaline conditions that can effectively cleave the uniquely introduced ester bonds. In order to further understand the structural and chemical features that may be associated with the observed improved processing efficiency, we used hyperspectral stimulated Raman scattering (hsSRS) to map the Zip-lignin in planta using lignin conjugated α–β carbon double bonds as the proxy. Such double bonds are introduced in all types of lignin, but unique structures in Zip-lignins experience a ~20 cm –1 shift from 1650 to 1630 cm –1 in their Raman spectra due to the conjugation between the double bond and an associated ester carbonyl group. Analysis of the hsSRS images has revealed that the Raman signal specifically representing Zip-lignin can be estimated. Moreover, it exhibits a distribution pattern in cell wall layers similar to that of the native lignin, but its intensity varies in different transgenic poplar lines. In addition, when the cell walls are imaged by atomic force microscopy and Simons’ staining, we found that the poplar containing the Zip-lignin had increased accessible areas, which may be beneficial to the chemical penetration during pretreatment and substrate accessibility during enzymatic hydrolysis. Our work indicates that both physical and chemical modification of cell wall in Zip-lignin poplar could contribute to the observed improvements in sugar yields.

09 BIOMASS FUELS↗
DOE OSTI2019

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT 50 , temperature that results in 50% injury of maximum, approximately -7 degrees C) and cold-acclimated (CA; leaf LT 50 approximately -50 degrees C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-a-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F 2 S 2 K n type; and RcDhn 2 as F 3 SK n type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

09 BIOMASS FUELS↗
DOE OSTI2018

Physical fractionation of sweet sorghum and forage/energy sorghum for optimal processing in a biorefinery

Sorghum offers great potential as a feedstock for the production of fuels and chemicals from both water-extractable sugars and the cell wall biopolymers, while its within-plant structural and compositional heterogeneity may allow for physical fractionations to tailor feedstock properties to a biorefining process. In this study, the stem internodes of two sorghum (Sorghum bicolor L. Moench) genotypes, a sweet sorghum (‘Della’) and a forage/energy sorghum (‘TX08001’), were first subjected to fractionation by manual classification by stem anatomy and internode proximity to the ground to yield 18 fractions. These fractions exhibited substantial differences in cell wall morphology, composition, and recalcitrance to mild alkaline pretreatment and enzymatic hydrolysis. While the sweet sorghum cultivar held nearly 70% more water-extractable sugar (sucrose, glucose, fructose, starch) in the stems than the forage/energy sorghum hybrid, both cultivars exhibited comparable diversity of composition and these compositions were remarkably similar in similar tissues and stem regions between the two cultivars. The fractions isolated from the pith parenchyma were the least recalcitrant to mild alkaline pretreatment and enzymatic hydrolysis and contained less lignin than fractions isolated from the epidermis, outer and inner rind, and internal vascular bundles. The pith samples isolated from the lowest region of the stem from both cultivars exhibited near-theoretical sugar hydrolysis yields when no pretreatment was employed and exhibited the lowest lignin contents of any of the fractions. Next, a physical fractionation approach approximating a commercial “de-pithing” process utilizing wet disintegration and sieving was applied to the forage/energy sorghum. A pith-rich fraction representing approximately 20% of the extractives-free mass of the stem could be isolated with this approach and, relative to the other fractions, was low in lignin, high in ash, highly hygroscopic, and showed an improved response to mild alkaline pretreatment and enzymatic hydrolysis at low enzyme loadings. Overall, these results demonstrate how heterogeneity within sorghum stems can be exploited using physical fractionation approaches to yield fractions enriched in desired properties that may allow for more streamlined processing.

09 BIOMASS FUELS↗
DOE OSTI2018

Co-Compartmentation of Terpene Biosynthesis and Storage via Synthetic Droplet

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield.

09 BIOMASS FUELS↗
DOE OSTI2015

Micro-Spectroscopic Imaging of Lignin-Carbohydrate Complexes in Plant Cell Walls and Their Migration During Biomass Pretreatment

In lignocellulosic biomass, lignin is the second most abundant biopolymer. In plant cell walls, lignin is associated with polysaccharides to form lignin-carbohydrate complexes (LCC). LCC have been considered to be a major factor that negatively affects the process of deconstructing biomass to simple sugars by cellulosic enzymes. Here, we report a micro-spectroscopic approach that combines fluorescence lifetime imaging microscopy and Stimulated Raman Scattering microscopy to probe in situ lignin concentration and conformation at each cell wall layer. This technique does not require extensive sample preparation or any external labels. Using poplar as a feedstock, for example, we observe variation of LCC in untreated tracheid poplar cell walls. The redistribution of LCC at tracheid poplar cell wall layers is also investigated when the chemical linkages between lignin and hemicellulose are cleaved during pretreatment. Our study would provide new insights into further improvement of the biomass pretreatment process.

09 BIOMASS FUELS↗
DOE OSTI2015

Study of Plant Cell Wall Polymers Affected by Metal Accumulation Using Stimulated Raman Scattering Microscopy

This project aims to employ newly-developed chemical imaging techniques to measure, in real-time, the concentration, dynamics and spatial distribution of plant cell wall polymers during biomass growth with inoculation of transgenic symbiotic fungi, and to explore a new pathway of delivering detoxified metal to plant apoplast using transgenic symbiotic fungi, which will enhance metal accumulation from soil, and potentially these metals may in turn be used as catalysts to improve the efficiency of biomass conversion to biofuels. The proposed new pathway of biomass production will: 1) benefit metal and radionuclide contaminant mobility in subsurface environments, and 2) potentially improve biomass production and process for bioenergy

09 BIOMASS FUELS↗