Vacuolar Localization of Proteases and Degradation of Chloroplasts in Mesophyll Protoplasts from Senescing Primary Wheat Leaves

Abstract

MesophyH protoplasts isolated from primary leaves of wheat seedlings were used to folow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at al stages of senescence. Al the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.Leaf senescence has often been referred to as a well-organized event (2, 4) because of the sequential loss of organelles with their corresponding function during senescence. Yet, evidence is lacking on how these changes might be controlled. If MATERIALS AND METHODSPlant Material. Wheat (Triticum aestivum L. var. Arthur) seeds were planted in vermiculite in 6-cm pots and held in a growth cabinet at 20°C and 75% RH. The photoperiod was 16 h, with a quantum flux density of 250 ,uE m s'. After 10 days, the second leaf had just emerged and the first leaf was fully expanded. The seedlings were then transferred to a dark room at 22°C to induce senescence. At different times, sections were taken from the primary leaves (8-cm sections cut 2 cm from the tip) and used for protoplast isolation.Protoplast, Chloroplast, and Vacuole Isolation. Protoplasts were isolated as previously described (6), except that 0.1% pectolyase Y-23 was substituted for prectinase and hemicellulase in the digestion enzyme mixture (5), which shortened the incubation time to 1 to 1.5 h. Chloroplasts were isolated according to the procedure of Robinson and Walker (10), as outlined previously (6).Vacuoles were isolated from protoplasts according to a modification of the method of Wagner (11). Twenty-eight ml of 0.5 mm DTT in 170 mm phosphate buffer (pH 8.0) were added to the centrifuge tube containing the protoplast pellet. The mixture was gently stirred for 5 min, poured through three layers of glasswool, and centrifuged at 200g for 3 min to spin down the majority of the protoplasts. The supernatant was transferred to a beaker, and 12 ml of 60o sucrose (w/v) were added to form an 18% sucrose mixture. The mixture was then divided into two centrifuge tubes, and 6 ml o...

Low-Temperature Damage and Subsequent Recovery of fab1 Mutant Arabidopsis Exposed to 2[deg]C

“…The changes we observed appear to represent a type of autophagy-a regulated breakdown of cytoplasmic and organellar components observed in leaf tissues and cultured cells during periods of induced starvation (Wittenbach, 1978;Wittenbach et al, 1982;Journet et al, 1986). The process of autophagy is reasoned to be a regulated metabolic response that permits cells and tissues to endure a prolonged period of starvation, with the best possible chances for survival and growth once favorable nutrient conditions are reestablished.…”

Programmed cell death during plant growth and development

“…The endoplasmic reticulum appears at least partially labile during senescence, while the nucleus appears to remain intact up to an advanced stage of chlorophyll loss (Thomson and Platt-Aloia, 1987). It is possible to isolate intact vacuoles from senescing leaves (Wittenbach et al, 1982). Moreover, that leaves remain turgid until advanced senescence provides indirect evidence for the persistence of an intact tonoplast (Smart, 1994).…”

“…In leaves, extensive cellular disorganization and cell death occur only after the majority of starch and protein have been remobilized (Wittenbach et al, 1982;Thomson and Platt-Aloia, 1987), suggesting that it may be possible to uncouple cell death from the remobilization phase of senescence. The ultrastructure, biochemistry and molecular biology related to remobilization have been extensively characterized.…”

“…Intact chloroplasts can be isolated from protoplasts prepared from dark-induced senescent wheat leaves from which up to 80% of the chlorophyll and protein have been lost (Wittenbach et al, 1982). Wittenbach et al (1982) suggested that autophagy of chloroplasts within the intact vacuole may be occurring during soybean leaf senescence.…”

“…Intact chloroplasts can be isolated from protoplasts prepared from dark-induced senescent wheat leaves from which up to 80% of the chlorophyll and protein have been lost (Wittenbach et al, 1982). Wittenbach et al (1982) suggested that autophagy of chloroplasts within the intact vacuole may be occurring during soybean leaf senescence. Respiratory activities of the mitochondria and catabolic activities of peroxisomes increase or remain steady during senescence (Smart, 1994) and mitochrondria remain intact.…”