Biology subjects

Walker, V. K.

Publications and source records attributed to Walker, V. K..

7 recordsLinked to original sources

bioRxiv2023

Characterization of skin- and intestine microbial communities in migrating high Arctic lake whitefish and cisco

At high latitudes, lake whitefish (Coregonus clupeaformis) and others in the closely related Coregonus species complex (CSC), including cisco (C. autumnalis and C. sardinella), can be diadromous, seasonally transitioning between freshwater lakes and the Arctic Ocean. CSC skin- and intestine microbiomes were collected, facilitated by Inuit fishers at sites on and around King William Island, Nunavut, at the northern range limits of lake whitefish. Community composition was explored using 16S rRNA gene sequencing, with significant differences in microbiota dispersions depending on fishing site salinity for lake whitefish intestine and skin, as well as cisco skin. Overall, lake whitefish intestine communities appeared more variable than cisco and had higher Shannon diversity, suggesting that lake whitefish and their microbiomes could be more susceptible to environmental stress possibly leading to dysbiosis. Although cisco condition was similar among distinct seasonal habitats, the higher average lake whitefish condition in freshwater rivers suggests that fishing these diadromous whitefish in estuaries may be optimal from a sustainable fishery perspective. Taken together, the impact of changing habitats on fish condition and different microbial composition may inform new approaches to CSC health in fisheries and aquaculture, in addition to being relevant for northern Indigenous peoples with subsistence and economic interests in these resources.

microbiology↗

bioRxiv2022

Chill injury in human kidney tubule cells after subzero storage is not mitigated by antifreeze protein addition

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to SCS. Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of subzero SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 {degrees}C) and at -6 {degrees}C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 {degrees}C, those stored at -6 {degrees}C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable subzero storage may not be effective for increasing storage times.

cell biology↗

bioRxiv2022

Whole genome sequencing of three mesorhizobia isolated from northern Canada to identify genomic adaptations promoting nodulation in cold climates

The N2-fixing symbiosis between rhizobia and legumes is negatively impacted by numerous stresses, including low temperatures. To identify genomic features and biochemical pathways of rhizobia that could foster improved symbiotic function under low temperatures, we isolated and characterized three Mesorhizobium strains from legume nodules collected at two distant northern Canadian sites. Whereas the classical determinants of nodulation and nitrogen fixation are located on the chromosome of most mesorhizobia, whole genome sequencing revealed that these genes are on a large symbiotic megaplasmid in all three of the newly isolated strains. A pangenome-wide association study identified 25 genes putatively associated with mesorhizobia isolated from arctic or subarctic environments, with the genomic location of many of these genes implying a relationship with legume symbiosis. Phylogenetic and sequence analyses of the common nodulation genes revealed alleles that are highly conserved amongst mesorhizobia isolated from northern climates but uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences and the horizontal transfer of these alleles between northern rhizobia. We speculate that nod sequence divergence was driven by climatic factors, and that the encoded proteins may be particularly stable and/or active at low temperatures.

microbiology↗

bioRxiv2022

Brachypodium antifreeze protein gene products inhibit ice recrystallization, attenuate ice nucleation, and reduce immune response

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, each Brachypodium AFP gene encodes two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models suggesting that ice-binding motifs on the ~13 kDa AFPs can "spoil" nucleating arrays on the ~120 kDa bacterial ice nucleating proteins that form ice at high sub-zero temperatures, consistent with decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Strikingly, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22), with structural modelling suggesting this was due to affinity of the LRR domains to flg22. Thus, these genes play distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins ability to adsorb to ice as well as attenuating bacterial ice nucleation and the host immune response.

plant biology↗

bioRxiv2022

Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freeze sensitivity

The model forage crop, Brachypodium distachyon, has a family of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were previously targeted for knockdown using a constitutive CaMV 35S promoter and the resulting transgenic Brachypodium showed reduced antifreeze activity and a greater susceptibility to freezing. However, the transgenic plants also showed developmental defects with shortened stem lengths and were almost completely sterile, raising the possibility that their reduced freeze tolerance could be attributed to developmental deficits. A cold-induced promoter from rice (prOsMYB1R35) has now been substituted for the constitutive promoter to generate temporal miRNA-mediated Brachypodium antifreeze protein knockdowns. Although transgenic lines showed no apparent pleiotropic developmental defects, they demonstrated reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, leaf infrared thermography, and leaf damage after infection with an ice nucleating phytopathogen. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8 {degrees}C was reduced by half or killed entirely, depending on the line, compared to cold-acclimated wild type plants. Although these proteins have been studied for almost 60 years, this is the first unequivocal demonstration in any organism of the utility of antifreeze protein function and their contribution to freeze protection, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from accessible cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced freezing tolerance.

plant biology↗

bioRxiv2021

The Brachypodium distachyon cold-acclimated plasma membrane proteome is primed for stress resistance

In order to survive sub-zero temperatures, some plants undergo cold acclimation where low, non-freezing temperatures and/or shortened day lengths allow cold hardening and survival during subsequent freeze events. Central to this response is the plasma membrane, where low-temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first plasma membrane proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time course experiment investigated cold acclimation-induced changes in the proteome following two-phase partitioning plasma membrane enrichment and label-free quantification by nano-liquid chromatography mass spectrophotometry. Two days of cold acclimation were sufficient for membrane protection as well as an initial increase in sugar levels, and coincided with a significant change in the abundance of 154 proteins. Prolonged cold acclimation resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained cold acclimation response elicited over several days. A meta-analysis revealed that the identified plasma membrane proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress and salt resistance suggesting crosstalk between stress responses, such that cold acclimation may prime plants for other abiotic and biotic stresses. The plasma membrane proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

plant biology↗

bioRxiv2018

baRcodeR with PyTrackDat: Open-source labelling and tracking of biological samples for repeatable science

Repeatable experiments with accurate data collection and reproducible analyses are fundamental to the scientific method but may be difficult to achieve in practice. Several flexible, open-source tools developed for the R and Python coding environments aid the reproducibility of data wrangling and analysis in scientific research. In contrast, analogous tools are generally lacking for earlier stages, such as systematic labelling and processing of field samples with hierarchical structure (e.g. time points of individuals from multiple lines or populations) or curating heterogenous data collected by different researchers over several years. Such tools are critical for modern research given trends toward globally distributed collaborators using higher-throughput technologies. As a step toward improving repeatability of methods for the collection of biological samples, and curation of biological data, we introduce the R package baRcodeR and the PyTrackDat pipeline in Python. The baRcodeR package provides tools for generating biologically informative, hierarchical labels with digitally encoded 2D barcodes that can be printed and scanned using low-cost commercial hardware. The PyTrackDat pipeline integrates with baRcodeR output to build a web interface for sample management and tracking along with data collection and curation. We briefly describe the application of principles from baRcodeR and PyTrackDat in three large research projects, which demonstrate their value to (i) help document sampling methods, (ii) facilitate collaboration and (iii) reduce opportunities for human errors and omissions that could otherwise propagate through downstream data analysis to compromise biological inference.

bioinformatics↗