Just like animals and human beings, plants too can be stressed. Whereas one may easily detect stress in animals and humans basing on their behavior, plant “behavior” is not easy to interpret.  

Why should we be concerned about stress in plants?

Plants under stress cannot reproduce or grow well because they have to divert much of their physiological energy to fighting stress. If the plants are critical food crops, this will lead to perpetual food shortages because their energy resources are spent fighting stress.

New study on measuring stress in plants

A new study titled “Sandwich Enzyme-Linked Immunosorbent Assay for Quantification of Callose” and led by a team of scientists at the Department of Plant Sciences, Microbiology and Biotechnology at Makerere University in collaboration with the University of California, Davis, USA, shows how stress in plants can be measured. According to Prof. Arthur Tugume, the lead scientist of this study and expert in plant diseases, when plants are stressed, they produce numerous molecules within their cells mostly as means of adapting to stress or avoiding the impact of stress. “For example, plants produce reactive oxygen species like hydrogen peroxide, superoxide ions, and hydroxyl ions. These reactive oxygen species are produced rapidly and act as rapid messengers in the plant tissues to activate additional responses spreading over the entire plant body. This helps the plant to withstand or avoid the impact of stress”.

Dr. Ssenku E. Jamilu, a plant physiology expert on the team explains that in plants, stress can be induced by many factors such as pollution in the soil and atmosphere, high soil salinity (salt stress), excessive lack of water (drought), extreme temperatures, lack of oxygen (anoxia), excess radiation, mechanical injury by wounding or by pests that feed on the plants, and infestation by disease-causing microorganisms (pathogens). “Such factors are worsened by climate change, implying the importance of measuring their impact on individual plants to guide plant breeding programmes to ensure sustainable food production in the face of climate change”.

The research indicates that reactive oxygen species set in motion additional processes to ensure limited impact of stress on the plant. For example, a unique carbohydrate, named “callose” starts to accumulate in large quantities within plant cells as a means of fortifying plant cells. Callose differs from the other usual plant carbohydrates such as starch or cellulose because of the way its structures are formed. Also, the production of callose gets increased during stress. Callose acts as a road-block to any pathogen such as bacteria by limiting bacterial movement that would otherwise ease attack on other tissues or cells.

 “If we can artificially induce the stress on the plant, and then measure the amount of reactive oxygen species or callose or any other responses in the plant, we should be able to directly and quantitatively measure the stress,” Prof. Tugume explains.

He however notes that callose participates in many other normal developmental processes of plants and for that reason, there is always some “housekeeping” callose in the plant tissues even without stress. “This means that one must be able to accurately and quantitatively distinguish between ‘stress-induced’ and normal ‘housekeeping’ callose”.

In this study, the researchers used young (2.5-months old) banana plantlets that had been generated from tissue culture at Kawanda Agricultural Research Institute. They then infected the plantlets with a bacterium called Xanthomonas campestris pv. musacearum. This bacterium induces so much stress on the banana plants resulting into a destructive disease known as banana xanthomonas wilt (BXW), the most destructive disease of bananas in East and Central Africa.

The banana leaves, pseudostems and corms were analyzed for callose and compared with the control plants that had been inoculated with water instead of bacteria. The analysis was done using a method called S-ELISA (Sandwich Enzyme-linked immunosorbent Assay), that was designed by scientists in this study. This method is specific to callose.

Callose-specific primary antibodies were used to coat the wells of 96-well microplates followed by incubation with callose extracts from tissues of stressed plants. Additional incubation with same primary callose-specific antibodies was done, and finally, incubation with a secondary antibody that is conjugated to an enzyme. When a substrate is supplied to the enzyme, colour changes were monitored. The presence of callose (and thus stress on the plants) was assessed quantitatively by measuring the intensity of color change resulting from enzymatic action on the substrate. This method proved effective in comparison to the gold standard method of detecting and quantifying aniline blue stained callose using fluorescence microscopy.

This research was part of the PhD studies for Mr. Abubakar S. Mustafa at Makerere University and University of California, Davis. According to Mr. Mustafa, his method is new and can be applied to any plant that is subjected to any stress because generally, plants produce callose in response to stress.” Mr. Mustafa further states that the method is convenient because, if necessary, samples can be processed between a few hours to one year (or even longer), which allows exchange of materials between laboratories and countries when needed, something that was not possible before. The S-ELISA method used 96-well plates which allows high throughout studies, hence, hundreds of samples can be analyzed simultaneously within a few days.

This study has been published as a Protocol by Multidisciplinary Publishing Institute (MDPI) in an open access journal, “Methods and Protocols”, in the section: Biochemical and Chemical Analysis & Synthesis and is freely accessible on https://www.mdpi.com/2409-9279/5/4/54/htm.

The research was funded by the Bill and Melinda Gates Foundation through the National Agricultural Research Organization (NARO), Uganda. The project had partners including the International Institute of Tropical Agriculture, the Alliance for Bioversity International and International Centre for Tropical Agriculture (CIAT) and CABI.

For more details, contact;

1. Prof. Arthur Tugume, Lead Scientist of the study, College of Natural Sciences, Makerere University, Email: arthur.tugume@mak.ac.ug, Tel: +256772514841
2. Mr. Abubakar S. Mustafa, Co-Author and PhD student on the study, Email: mustafa.abubakar.sadik@gmail.com, Tel: +256702813233
3. Hasifa Kabejja, Principal Communication Officer, College of Natural Sciences, Makerere University, Email: pr.cns@mak.ac.ug, Tel: +256774904211

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