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Development of self-burrowing probes that can penetrate soils without the aid of external reaction force from drill rigs and trucks would facilitate site characterization activities and deployment of sensors underneath existing structures and in locations with limited access (e.g., toe of dams, extraterrestrial bodies). Successful deployment of self-burrowing probes in the field will require several cycles of expansion, penetration, and contraction motions due to the geometric constraints and the increase in soil strength with depth. This study explores the multi-cycle performance of a dual-anchor self-burrowing probe in granular assemblies of varying density using discrete element modeling simulations. The simulated probe consists of an expandable top shaft, expandable bottom shaft, and a conical tip. The expansion of the shafts are force-controlled, the shaft contraction and tip advancement are displacement-controlled, and the horizontal tip oscillation is employed to reduce the penetration resistance. The performance of the self-burrowing probe in terms of self-burrowing distance is greater in the medium dense specimen than in the dense and loose specimens due to the high magnitude of anchorage force in comparison with penetration resistance. For all three soil densities, most of the mechanical work is done by tip oscillation; however, this accounts for a greater percentage of the total work in the denser specimen. Additionally, while tip oscillation aids in enabling self-burrowing to greater depths, it also produces a greater work demand. The results presented here can help evaluate the effects of soil density on probe prototypes and estimate the work requited for self-burrowing.

 

Abstract Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops. 

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

 

Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)‐O‐glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp‐O‐glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of “Ser–Hyp–Hyp–Hyp–Hyp” motif or (SP4)₁₈was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) fromAcidothermus cellulolyticus. The engineered (SP4)₁₈module was extensively Hyp‐O‐glycosylated with arabino‐oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5–11‐fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)₁₈module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)₁₈‐tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5‐fold without having adverse impact on the plant growth.

 

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGPmodule secreted up to 56‐fold greater levels ofEGFP, compared with anEGFPcontrol lacking any HypGPmodule, supporting the function of HypGPmodules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈and (SP)₃₂modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fusedEGFPprotein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFPand (SP)₃₂‐EGFPintermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGPmodules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈module andAGP‐styled (SP)₃₂module is proposed.