Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy.

Abstract

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function.We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A-gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes inArabidopsisHypocotyls during Seedling Growth

“…The transition of seedling peroxisomes to leaf peroxisomes appears to be a gradual process, as several reports described a transitional form of peroxisomes containing both glyoxylate cycle and photorespiratory enzymes (Titus and Becker, 1985;Nishimura et al, 1986;Sautter, 1986). We do not know which of these three types of peroxisomes is subjected to autophagy ( Figure 5).…”

“…As a control, we compared the expression pattern of ATG in cotyledons (see Supplemental Figure 5 online). We focused on genes encoding the ATG8 conjugation system because the expression of these genes is often regulated developmentally (van der Graaff et al, 2006;Chung et al, 2009). A preliminary search in the Genevestigator database (http://www.genevestigator.com) revealed that ATG3, ATG8b, ATG8g, and ATG8i showed higher expression levels in hypocotyls than in cotyledons.…”

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

“…Immunolabeling experiments in greening cucurbit cotyledons directly demonstrate the presence of both photorespiration enzymes and glyoxylate cycle enzymes in the same peroxisomes (Titus and Becker, 1985;Nishimura et al, 1986;Sautter, 1986). Moreover, MLS is stable following in vitro import into early pumpkin (Cucurbita sp Amakuri Nankin) seedling peroxisomes but degraded after import into transitional peroxisomes (Mori and Nishimura, 1989).…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…During seedling development, peroxisome functions shift from fatty acid utilization to photorespiration (Titus and Becker, 1985;Nishimura et al, 1986;Sautter, 1986;. Autophagy mutants accumulate peroxisomal proteins (Shibata et al, 2013;Yoshimoto et al, 2014) and peroxisomes Yoshimoto et al, 2014) during this transition, suggesting a role for pexophagy in clearing obsolete peroxisomes.…”

“…In addition to the core processes of b-oxidation and ROS detoxification, plant peroxisomes house diverse specialized functions (for review, see Reumann and Bartel, 2016) that may change during development (Titus and Becker, 1985;Nishimura et al, 1986;Sautter, 1986; or in response to environmental cues (for review, see Goto-Yamada et al, 2015). For example, plant peroxisomes sequester enzymes acting in photorespiration, which is important when ribulose-1,5-bisphosphate carboxylase/oxygenase fixes O 2 instead of CO 2 .…”