SummaryA rice suspension cell culture system has been established to study how sugar depletion regulates (x-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa (x-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of (xamylases of 46 kDa is dramatically induced when cells are starved of sucrose~Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplaats. Immunocytochemical studies revealed that (xamylases are localized in starch granules within amyloplests, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice (x-amylases suggests hitherto unidentified pathways for import of (xamylases into amyloplaats. The studies show that differential (x-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of (x-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of (x-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.
a-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether a-amylases are localized in plastids where starch is stored. To study the subcellular localization of a-amylases in plant cells, a rice (Oryza sativa) a-amylase, aAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of aAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated aAmy3 to be cotranslationally imported and processed in canine microsomes. aAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with M r similar to aAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized aAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the aAmy3 SP was further studied by gain-of-function analyses. b-Glucuronidase (GUS) and green fluorescence protein fused with or without the aAmy3 SP was expressed in transgenic tobacco or rice. The aAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice a-amylase, aAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.Two types of starch exist in plants: transitory (assimilatory) starch, which is located in chloroplasts, and reserve starch, which is deposited in amyloplasts. Degradation of starch can be either hydrolytic, mainly catalyzed by a-amylase, b-amylase, and debranching enzymes, or phosphorolytic, catalyzed by starch phosphorylase. Degradation of reserve starch in cereal grains is mainly hydrolytic, whereas degradation of transitory starch in leaves can be hydrolytic and/or phosphorolytic (Beck and Ziegler, 1989). In germinating cereal grains, a-amylases are the most abundant starch-degrading enzymes. The enzymes are secreted by aleurone cells into the starchy endosperm where they degrade the starch grains (Jacobsen et al., 1995). Whether a-amylases also play an essential role in starch degradation in photosynthetic tissues and in tissues other than endosperm is not clear. In most plants, starch can be found in pollens, seeds, leaves, stems, roots, and other tissues. In spinach (Spinacia oleracea), a-amylases are the only enzymes that have been demonstrated to attack starch granules isolated from chloroplasts (Steup et al., 1983).Determination of the subcellular distribution of a-amylases is essential for understanding the physiological function of these enzymes in...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.