Confocal fluorescence microscopy investigation for the existence of subdomains within protein storage vacuoles in soybean cotyledons

Authors: Krishnan, Hari; JURKEVICH, ALEXANDER - University Of Missouri

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2022
Publication Date: 3/27/2022
Citation: Krishnan, H.B., Jurkevich, A. 2022. Confocal fluorescence microscopy investigation for the existence of subdomains within protein storage vacuoles in soybean cotyledons. International Journal of Molecular Sciences. 23(7). Article 3664. https://doi.org/10.3390/ijms23073664.
DOI: https://doi.org/10.3390/ijms23073664

Interpretive Summary: Soybeans contain about 35-40% protein on a dry weight basis. The two most abundant groups of proteins found in soybean seeds are called the 11S and 7S globulins. These two groups of proteins accumulate during seed development and are stored in special structures named protein storage vacuoles. In several plants, different type of proteins, such as 7S and 11S globulins, are stored in separate regions (subdomains) within the protein storage vacuoles. In this basic study, the presence or absence of subdomains within protein storage vacuoles was investigated with the aid of confocal fluorescent microscopy. Our study revealed the presence of potential subdomains with soybean protein storage vacuoles when the seeds were chemically fixed for microscopy. However, fixation independent studies have shown that these subdomains are chemical fixation artifacts. Our results offer new knowledge on the accumulation of soybean seed proteins. A better understanding of protein accumulation will enable scientists the ability to manipulate the quality and quantity of soybean seed protein, which will benefit US soybean farmers.

Technical Abstract: In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.