Gene Expression Patterns, Localization, and Substrates of Polyphenol Oxidase in Red Clover (Trifolium pratense L.)

Abstract

Polyphenol oxidase (PPO) genes and their corresponding enzyme activities occur in many plants; natural PPO substrates and enzyme/substrate localization are less well characterized. Leaf and root PPO activities in Arabidopsis and five legumes were compared with those of high-PPO red clover ( Trifolium pratense L.). Red clover PPO enzyme activity decreased leaves > stem > nodules > peduncle = petiole > embryo; PPO1 and PPO4 genes were expressed early in leaf emergence, whereas PPO4 and PPO5 predominated in mature leaves. PPO1 was expressed in embryos and nodules. PPO substrates, phaselic acid and clovamide, were detected in leaves, and clovamide was detected in nodules. Phaselic acid and clovamide, along with caffeic and chlorogenic acids, were suitable substrates for PPO1, PPO4, and PPO5 genes expressed in alfalfa ( Medicago sativa L.) leaves. PPO enzyme presence and activity were colocalized in leaves and nodules by cytochemistry. Substrates and PPO activity were localized in developing squashed cell layer of nodules, suggesting PPO may have a developmental role in nodules.

Phenolic Profiles and Polyphenol Oxidase (PPO) Gene Expression of Red Clover (<i>Trifolium pratense</i>) Selected for Decreased Postharvest Browning

“…However, monitoring expression of PPO1 and PPO3 may not have adequately captured PPO variation among selection cycles because it is now known that red clover has about six PPO genes [33]. The PPO4 gene, which was identified by Winters et al [33], was expressed more strongly than PPO1 in mature leaves of red clover [10]. Given the maturity of the red clover at harvest, it is possible that PPO4 expression would have been stronger than PPO1 or PPO3 expression, and more responsive to selection for decreased browning.…”

“…However, given the lack of change in PPO1 gene expression, the observed decrease in clovamide, which can bind to proteins in the presence or absence of PPO1 [9], may indicate that decreased browning has the greatest effect on concentrations of phenolics that can bind protein independently of PPO1 activity. Dependence on PPO4 activity cannot be ruled out because PPO4 can utilize clovamide as a substrate [10]. Potential effects of a decrease in sissotrin (biochanin A glucoside) are difficult to assess because total biochanin A did not decrease in Cycle 8.…”

“…These latter two compounds are present primarily as malonylglucosides (Figure 1(b)) in planta [5]- [7]. Two dominant caffeic acid derivatives are phaselic acid and clovamide (Figure 1(c)) [8]- [10]. These are o-diphenols (compounds with hydroxy groups ortho to each other on the benzene ring).…”

Two New Polyphenol Oxidase Genes of Tea Plant (Camellia sinensis) Respond Differentially to the Regurgitant of Tea Geometrid, Ectropis obliqua

“…tomato and to Spodoptera litura (Fabricius) [11,17,18]. In addition, PPO genes are frequently found to be differentially induced in response to injuries inflicted by wounding, pathogens, or herbivores from various plant species, and also to signaling molecules (jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), ethylene (ET)), suggesting that these genes have a defensive role [19,20,21,22,23]. …”

Detection of Potential Chloroplastic Substrates for Polyphenol Oxidase Suggests a Role in Undamaged Leaves

“…Red clover has been extensively used to study PPO activity (Winters et al, 2003; Sullivan et al, 2004; Lee et al, 2010; Webb et al, 2013). Phaselic acid and clovamide are the main endogenous o -diphenol substrates detected in red clover leaves (Winters et al, 2008; Sullivan, 2009).…”