Comparing Methods for Detection and Quantification of Plasmodesmal Callose in Nicotiana benthamiana Leaves During Defense Responses.

Callose, a ß-(1,3)-d-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD or, conversely, by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing likely intercellular trafficking activity during plant immunity. Despite the popularity of this metric, there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying plasmodesmal callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescence microscopy to measure callose deposition in fixed tissue. Manual or semiautomated workflows for image analysis were also compared and found to produce similar results, although the semiautomated workflow produced a wider distribution of data points. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Reliable detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses.

Callose, a beta-(1,3)-D-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD, or conversely by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during innate immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing the intercellular trafficking activity during plant immunity. Despite the popularity of this metric there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying PD callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescent microscopy to measure callose deposition in fixed tissue. Manual or semi-automated workflows for image analysis were also compared and found to produce similar results although the semi-automated workflow produced a wider distribution of data points.

Approaches for investigating plasmodesmata and effective communication.

Plasmodesmata (PD) are integral plant cell wall components that provide routes for intercellular communication, signaling, and resource sharing. They are therefore essential for plant growth and survival. Much effort has been put forth to understand how PD are generated and their structure is refined for function and to determine how they regulate intercellular trafficking. This review provides an overview of some of the approaches that have been used to study PD structure and function, highlighting those that may be more widely adopted to address questions of PD cell biology and function. Extending our focus on the importance of communication, we address how effective communication strategies can increase diversity and accessibility in the research laboratory, focusing on challenges faced by our deaf/hard-of-hearing colleagues, and highlight successful approaches to including them in the research laboratory.

Chloroplast-to-nucleus retrograde signalling controls intercellular trafficking via plasmodesmata formation.

The signalling pathways that regulate intercellular trafficking via plasmodesmata (PD) remain largely unknown. Analyses of mutants with defects in intercellular trafficking led to the hypothesis that chloroplasts are important for controlling PD, probably by retrograde signalling to the nucleus to regulate expression of genes that influence PD formation and function, an idea encapsulated in the organelle-nucleus-PD signalling (ONPS) hypothesis. ONPS is supported by findings that point to chloroplast redox state as also modulating PD. Here, we have attempted to further elucidate details of ONPS. Through reverse genetics, expression of select nucleus-encoded genes with known or predicted roles in chloroplast gene expression was knocked down, and the effects on intercellular trafficking were then assessed. Silencing most genes resulted in chlorosis, and the expression of several photosynthesis and tetrapyrrole biosynthesis associated nuclear genes was repressed in all silenced plants. PD-mediated intercellular trafficking was changed in the silenced plants, consistent with predictions of the ONPS hypothesis. One striking observation, best exemplified by silencing the PNPase homologues, was that the degree of chlorosis of silenced leaves was not correlated with the capacity for intercellular trafficking. Finally, we measured the distribution of PD in silenced leaves and found that intercellular trafficking was positively correlated with the numbers of PD. Together, these results not only provide further support for ONPS but also point to a genetic mechanism for PD formation, clarifying a longstanding question about PD and intercellular trafficking. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Local site variation in stopover physiology of migrating songbirds near the south shore of Lake Ontario is linked to fruit availability and quality.

Birds that migrate long distances between breeding and wintering grounds are challenged to find adequate stopover sites that can provide a high-quality source of nutrition in order to refuel quickly and continue on their migratory journeys. Wild fruits are a well-documented component in the diets of many passerines during autumn migration. Thus, fruit availability and the proliferation of shrubs that bear low-quality fruits at important stopover sites may dictate the quality of food resources available for refuelling birds and present a conservation concern. We profiled plasma metabolites of two migratory passerine species at two different stopover sites near the south shore of Lake Ontario during the peak of autumn migration. We also measured diversity, availability and nutritional quality of fruits present at these sites. Site explained most of the variation in plasma triglyceride for both bird species, but was less important than other confounding variables for explaining concentrations of plasma ß-hydroxybutyrate and plasma uric acid concentrations. Site differences in fat deposition, as indicated by plasma triglyceride, may in part be explained by the large differences in diversity and availability of high-quality fruits between the two sites. Our results suggest that abundant, lipid-rich native fruits with high-energy density are associated with increased fat deposition during autumn stopovers for some species, although other factors, such as proximity to the Lake Ontario shoreline and the opportunities to refuel in the surrounding landscape, are likely to play a role in stopover site use by birds. It is possible that local site characteristics that influence growing conditions may impact the quality of fruits produced by a plant species, altering the availability of critical nutrients for avian consumers.