Premise: Fluorescence microscopy is an effective tool for viewing plant internal anatomy. However, using fluorescent antibodies or labels hinders throughput. We present a minimal protocol that takes advantage of inherent autofluorescence and aldehyde‐induced fluorescence in plant cellular and subcellular structures to markedly increase throughput in cellular and ultrastructural visualization.Methods and Results: Twelve species distributed across the plant phylogeny were each subjected to five fixative treatments: 1% paraformaldehyde and 2% glutaraldehyde, 2% paraformaldehyde, 2% glutaraldehyde, formalin‐acid‐alcohol (FAA), and 70% ethanol. Samples were prepared by embedding and mechanically sectioning or via whole mount. A confocal laser scanning system was used to collect micrographs. We evaluated and compared fixative influence on sample structural preservation and tissue autofluorescence.Conclusions: Formaldehyde fixation of Viridiplantae taxa samples generates useful structural data while requiring no additional histological staining or clearing. In addition, a fluorescence‐capable microscope is the only specialized equipment required for image acquisition. The minimal protocol developed in this experiment enables high‐throughput sample processing by eliminating the need for multi‐day preparations.

Understanding plant adaptation mechanisms to prolonged water immersion provides options for genetic modification of existing crops to create cultivars more tolerant of periodic flooding. An important advancement in understanding flooding adaptation would be to elucidate mechanisms, such as aerenchyma air-space formation induced by hypoxic conditions, consistent with prolonged immersion. Lysigenous aerenchyma formation occurs through programmed cell death (PCD), which may entail the chemical modification of polysaccharides in root tissue cell walls. We investigated if a relationship exists between modification of pectic polysaccharides through de-methyl esterification (DME) and the formation of root aerenchyma in select Fabaceae species. To test this hypothesis, we first characterized the progression of aerenchyma formation within the vascular stele of three different legumes—Pisum sativum, Cicer arietinum, and Phaseolus coccineus—through traditional light microscopy histological staining and scanning electron microscopy. We assessed alterations in stele morphology, cavity dimensions, and cell wall chemistry. Then we conducted an immunolabeling protocol to detect specific degrees of DME among species during a 48-hour flooding time series. Additionally, we performed an enzymatic pretreatment to remove select cell wall polymers prior to immunolabeling for DME pectins. We were able to determine that all species possessed similar aerenchyma formation mechanisms that begin with degradation of root vascular stele metaxylem cells. Immunolabeling results demonstrated DME occurs prior to aerenchyma formation and prepares vascular tissues for the beginning of cavity formation in flooded roots. Furthermore, enzymatic pretreatment demonstrated that removal of cellulose and select hemicellulosic carbohydrates unmasks additional antigen binding sites for DME pectin antibodies. These results suggest that additional carbohydrate modification may be required to permit DME and subsequent enzyme activity to form aerenchyma. By providing a greater understanding of cell wall pectin remodeling among legume species, we encourage further investigation into the mechanism of carbohydrate modifications during aerenchyma formation and possible avenues for flood-tolerance improvement of legume crops.

Flooding can cause root hypoxia and can lead to significant agricultural losses. Therefore, understanding plant adaptations to flooding, including root aerenchyma development, is one important avenue for insuring future global food security. We investigated cell wall modifications during root aerenchyma formation in response to the prolonged 0–48 h flooding of Phaseolus coccineus, Pisum sativum, and Cicer arietinum seedlings. Using transmission electron microscopy, toluidine blue O (TBO) staining, and immunolabeling with antibodies targeting de-methyl-esterified homogalacturonan (DMEH), partially DMEH, and methyl-esterified homogalacturonan (MEH), we examined changes in cell wall composition. Transmission electron microscopy and TBO staining revealed degradation of cell walls and middle lamella, with accumulation of DMEH near flooding-induced aerenchyma cavities. Immunolabeling indicated increased DMEH epitope availability in flooded roots, suggesting a role in cell wall remodeling. Enzyme pretreatments, used to “unmask” homogalacturonan by removing cellulose and hemicellulose, revealed that specific forms of homogalacturonan, particularly DMEH complexed with calcium and MEH, are masked by these cell wall components. The study highlights the complex interplay of pectin, cellulose, and hemicellulose in cell wall degradation during aerenchyma development, providing insights into legume flooding stress responses.

Characterizing the extracellular mechanisms plants use to adapt to water immersion could provide avenues for crop improvement in the face of periodic flooding. One such mechanism is hypoxia-induced aerenchyma formation. The formation of lysigenous aerenchyma occurs through programmed cell death (PCD) that may require the chemical modification of select polysaccharides in root cell walls. Currently, the precise mechanism of cell wall structural modification is not fully defined. To better characterize this mechanism, we investigated whether a relationship exists between modification of cell wall xyloglucans through removal of fucosyl functional groups and the formation of root aerenchyma in agriculturally relevant Fabaceae species. Characterization was conducted through the immunolabeling of specific xyloglucan epitopes within root cell walls during a 48-hour flooding time series. Immunolabeling results suggest progressive alterations in the accessibility or abundance of fucosylated and non-fucosylated xyloglucan epitopes in cell walls bordering developing aerenchyma cavities during PCD. Additionally, we performed an enzymatic pretreatment to remove select cell wall polymers prior to immunolabeling for xyloglucan, xylan and crystalline cellulose. These pretreatments demonstrate potential in vitro epitope masking constraints in root cell walls near developing aerenchyma. Our findings suggest that chemical modification to xyloglucans occurs in tandem with modification of select cell wall pectin epitopes. Our results elucidate previously uncharacterized cell wall carbohydrate remodeling during PCD leading to aerenchyma formation among legume species.