Azure c600 used to detect fluorescence-based proanthocyanidins in situ

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Proanthocyanidins (PAs) are found in plants and have a role in protecting plants against herbivores and fungal pathogens. They are also used in many industrial applications due to the importance of PAs’ function and dynamics in the plant cell walls; however, there is a lack of sufficient methods to analyze them in planta. A research team at the Swedish University of Agricultural Sciences found 4-dimethylaminocinnamaldehyde (DMACA) can be used as a PA-specific fluorescent dye to localize PAs in plant cells1. The Azure c600 Western blot imager served an important role in the study, as the team used it to visualize and validate using DMACA to localize PAs in leaves. Prior to this study, how PAs incorporate into plant cell walls or what their functions under stress conditions were, was unknown.

Since the release of this publication, the cSeries Imaging Systems have been succeeded by the new Azure Imaging Systems. The upgraded systems are high performance instruments capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

Fluorescent detection method for PAs

To understand the dynamics of PAs in cell walls, high-resolution microscopy is required, along with fluorescent dyes that can identify PAs. Common methods for detecting PAs in plants requires maceration of the plant tissue for processing, ruling out in situ methods. Chowdhury et al set out to identify a PA-specific fluorescent dye that could be used for in situ analysis of PAs. DMACA has been used before in light microscopy to identify PAs as its reaction with PA causes a blue precipitate, but the use of DMACA as a PA-specific fluorophore for high-resolution microscopy had not been evaluated. Chowdhury and team explored this possibility.

More Reading: Problems With Ponceau Stain? Consider Alternative Total Protein Stains for Fluorescent Western Blots

Investigating PA-specific fluorogenic properties of DMACA

Using commercially available PAs and isolated PAs from poplar roots, the researchers tested if DMACA would fluoresce in a PA-specific manner. Their results showed classic DMACA excitation and emission was only observed in samples containing PAs. With the confirmation that DMACA is PA-specific and fluoresces, they began to look for any potential interference from proteins found in the plant cell wall.

The researchers investigated whether DMACA fluoresces in the presence of isolated cell wall polymers, such as cellulose. To evaluate this, they implemented the fluorescence spot test (FST) method. PAs and cell wall polysaccharides were mixed with the DMACA reagent and before being added to a PVDF membrane. An Azure c600 was used to acquire images using three RGB channels: Cy2, Cy3, and Cy5 (Figure 2).

The Ultimate Western Blot Imaging System

The Azure 600 offers laser technology with two IR detection channels enabling you to image more than one protein in an assay. It provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.
Scientist choosing settings on Azure 600
The Azure 600 is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.

Chowhury et al confirmed their previous notion of the existence of a PA-dependent shift from Cy3 to Cy5 with DMACA, where the fluorescence intensity was dependent on PA concentration. Using the semi-quantitative FST method, we now know plant polymers do not interfere with PA-specific DMACA fluorescence in the Cy 5 channel. Chlorophyll, however, proved troublesome, since the emission range of PA-specific DMACA overlaps with the chlorophyll’s autofluorescence.

Results of the fluorescence spot test using Azure c600 Western blot imager
Figure 2 from Chowhury et al 2022 representing the results of the fluorescence spot test (FST) method. The DMACA fluorescence with different PA species and cell wall polysaccharides at various dilutions. Fluorescence was detected with the Azure c600 imager.

After using ethanol to remove chlorophyll, the researchers used the Azure c600 to examine the blue precipitate resulting from DMACA-PA interaction in leaves under both white light and fluorescence (Figure 3). Overall, the fluorescence corresponded to the areas with the blue precipitate, successfully indicating DMACA could be used to detect PAs in planta.

Bright light and fluorescent images of leaves exposed to DMACA
Figure 3 from Chowdhury et al. 2022. The bright light and fluorescent images of leaves exposed to DMACA captured using Azure c600. The blue precipitate shows DMACA interacting with PAs (D) and the red fluorescent areas (E) overlap these blue areas.

PA-specific DMACA used to study PAs in situ in plant roots

Chowdhury and team then looked at DMACA for use in fluorescence microscopy for in situ analysis of PAs in root tissue. Under bright light microscopy, the DMACA-PA blue precipitate reaction method was used once more to locate the PAs exact location in the root tip. The team found fluorescence overlapped with blue precipitate areas, indicating the presence of PAs in the roots. In the brightfield images, their findings showed fluorescence was more prominent than the blue coloration; DMACA fluorescence is a sensitive way to visualize cell-wall bound PAs. The team concluded PA-specific DMACA signal both is highly stable and, while it can be useful in leaves, more suitable for use in roots.

Upon proving the ability of DMACA to serve as a fluorophore specific for PAs, further investigation of the co-localization of PAs with cell wall polymers was needed. PAs overlapped significantly with other cell wall polymers in older portions of the root, but were absent from the newly formed root tip. The results from this paper indicate PAs are incorporated later in development.

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What are PAs and why are they important?

PAs are commonly found in woody plants, such as grapes, and forest trees. They are an end product of the flavonoid biosynthetic pathway and act as plant pigments and may prevent cancer2. PAs are involved in plant defense and protect plants by acting against leaf-eating herbivores, fungal pathogens, and UV damage.

Takeaways from this study

In this groundbreaking study, Chowdhury et al was able to characterize the fluorogenic properties of DMACA as a PA-specific fluorophore and validate its use in fluorescence microscopy as they studied PAs in situ and in planta. The team identified a novel tool which can be used to study PAs and their dynamics at the subcellular level. This study opens the door for further study of PAs in plants.

SOURCES

  1. Chowdhury J, Ferdous J, Lihavainen J, Albrectsen BR, Lundberg-Felten J. Fluorogenic properties of 4-dimethylaminocinnamaldehyde (DMACA) enable high resolution imaging of cell-wall-bound proanthocyanidins in plant root tissues. Front Plant Sci. 2023 Jan 16;13:1060804. doi: 10.3389/fpls.2022.1060804. PMID: 36726681; PMCID: PMC9884812.
  2. “Proanthocyanidins – Health Encyclopedia – University of Rochester Medical Center.” URMC, https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=19&contentid=proanthocyanidins. Accessed 11 March 2023.

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