Polyphenol oxidase affects normal nodule development in red clover (Trifolium pratense L.)

Authors: WEBB, K - Aberystwyth University; COOKSON, ALAN - Aberystwyth University; ALLISON, GORDON - Aberystwyth University; Sullivan, Michael; WINTERS, ANA - Aberystwyth University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2014
Publication Date: 11/24/2014
Publication URL: https://handle.nal.usda.gov/10113/60084
Citation: Webb, K.J., Cookson, A., Allison, G., Sullivan, M.L., Winters, A.L. 2014. Polyphenol oxidase affects normal nodule development in red clover (Trifolium pratense L.). Frontiers in Plant Science. 5:700. doi: 10.3389/fpls.2014.00700.

Interpretive Summary: Polyphenol oxidases (PPOs) are enzymes present in nearly all plants. PPOs oxidize o-diphenolic compounds present in plants to o-quinones. These quinones are highly reactive and undergo a number of secondary chemical reactions. These secondary reactions are most widely associated with post-harvest browning in fruits, vegetables, and other agricultural crops. Quinone reactions resulting from the action of PPO have also been shown by ARS scientists to protect forage protein during storage. Despite their presence in a wide variety of plant species, relatively little is known about what roles PPO enzymes play in normal plant growth and development. In this study, red clover plants in which PPO activity had been suppressed via the molecular biology technique of gene silencing were compared to red clover plants with normal levels of PPO. This was done in order to gain insights into the possible roles PPOs play in normal development of plants, and especially in nodules, the special nitrogen-fixing structures that develop on roots of legumes like red clover. Suppression of PPO resulted in major changes to the oxido-reduction status of plants, which could lead to susceptibility of plants to certain stresses. Further, while lack of PPO in the suppressed red clover plants does not have an obvious effect on plants grown under optimal, stress-free nitrogen-fixation conditions, the nitrogen-fixing nodules were significantly altered (biochemistry, chemical composition, and developing morphology/anatomy). This suggests, at least in red clover, that PPO may play a significant role in the important process of nitrogen fixation. Future studies will attempt to define the role PPO plays. The findings of this work will be useful to other research scientists interested in plant responses to stress, symbiotic nitrogen fixation, and PPO enzymes in general.

Technical Abstract: Polyphenol oxidase (PPO) may have multiple functions in tissues, depending on its cellular or tissue localization. We used PPO RNAi transformants of red clover (Trifolium pratense) to determine the role PPO plays in normal development of plants, and especially in nitrogen-fixing nodules. In red clover, PPO was not essential for either growth or nodule production, or for nodule function in plants grown under optimal, N-free conditions. However, absence of PPO resulted in a more reduced environment in all tissues, as measured by redox potential, and caused subtle developmental changes in nodules. Leaves (and to a lesser extent nodules) lacking PPO tended to accumulate phenolic compounds. A comparison of nodules of two contrasting clones by microscopy revealed that nodules lacking PPO were morphologically and anatomically subtly altered, and that phenolics accumulated in different cells and tissues. Developing nodules lacking PPO were longer, and there were more cell layers within the squashed cell layer (SCL). However, the walls of these cells were less thickened and the cells were less squashed. Within the N2-fixing zone, bacteroids appeared more granular and were less tightly packed together.