Changes in lipoxygenase isoforms during germination and early seedling growth of Pisum sativum L.

Abstract

Investigations of physiological functions of seed lipoxygenase during germination and early seedling growth are complicated by the presence of multiple isoforms with different product specificity. The combination of isoelectric focusing, lipoxygenase in-gel activity staining and mass spectrometry showed that only two seed lipoxygenases, LOX-2 and LOX-3, were active at neutral pH in protein extracts from dry and germinating pea seeds (Pisum sativum L.), with LOX-3 more abundant than LOX-2. Each active seed LOX appeared at multiple pIs, probably resulting from post-translational modifications. The abundance of pea seed lipoxygenases was lower in embryonic shoots than in other parts of the axis. After radicle emergence, seed LOXs were degraded, as revealed by Western blotting using antibodies specific to pea seed LOXs. Two new vegetative isoforms, LOX-N3 and LOX-g, appeared in the shoot and gradually replaced the pea seed lipoxygenases as the principal contributors to LOX enzyme activity during early seedling growth. Activities of LOX-N3 and LOX-g were abundant in shoot tips, but not root tips, in pea seedlings. Thus, during germination, there is a shift of LOX activity from radicles to shoots that accompanies the transition from seed LOXs to vegetative LOXs. These results confirmed that identification of pea LOXs based solely on pI may be misleading; however, mass spectrometry enabled more accurate identification of these highly homologous proteins to be made.

Relationship between proportion and composition of albumins, and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.)

“…The multiple SDS‐PAGE bands around 100 kDa are probably lipoxygenase isoforms, including LOX‐2 (97 kDa) and LOX‐3 (100 kDa) 29. Although several previous studies reported the existence of lipoxygenase isoforms,27, 29 the quantitative variation in lipoxygenase among current cultivated pea varieties was first identified in this study. Lifter, which was highest in seed protein content among the seven smooth pea varieties, however, exhibited comparable density of the lipoxygenase band, but distinctively lower density of the PA2 band (around 30 kDa) than other varieties.…”

“…On the other hand, Shawnee, which was relatively high in seed protein content and low in albumin:globulin ratio (see Tables 1 and 2), exhibited the lowest lipoxygenase intensity (Table 3). The multiple SDS‐PAGE bands around 100 kDa are probably lipoxygenase isoforms, including LOX‐2 (97 kDa) and LOX‐3 (100 kDa) 29. Although several previous studies reported the existence of lipoxygenase isoforms,27, 29 the quantitative variation in lipoxygenase among current cultivated pea varieties was first identified in this study.…”

Understanding the role of H2O2 during pea seed germination: a combined proteomic and hormone profiling approach

“…These enzymes catalyze the hydroperoxidation of polyunsaturated fatty acids to produce unsaturated fatty acid hydroperoxides. The hydroperoxides are subsequently metabolized via secondary pathways to form bioactive compounds (Mo & Koster 2006). Pea seed LOXs are characterized by two major isoforms.…”

Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation

“…LOXs, belonging to the nonheme ironcontaining dioxygenases, catalyze the addition of molecular oxygen to polyunsaturated fatty acids containing a (Z,Z)-1,4-pentadiene system to produce an unsaturated fatty acid hydroperoxide (Porta and Rocha-Sosa, 2002). In seeds, LOXs may function as storage proteins, assist membrane retailoring during germination, and play a role in pathogen resistance (Porta and Rocha-Sosa, 2002;Mo and Koster, 2006). In addition, LOXs have been observed to localize to the oil bodies (Feussner et al, 2001;Zienkiewicz et al, 2014) and are thought to play an important role in regulating lipid degradation during seed germination (Feussner et al, 2001;Rudolph et al, 2011).…”