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Arabidopsis V-ATPase B Subunits Are Involved In Dynamic Actin Cytoskeleton Remodeling

Posted on:2012-09-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:B Y MaFull Text:PDF
GTID:1100330335966560Subject:Botany
Abstract/Summary:PDF Full Text Request
Vacuolar-type H+-ATPase (V-ATPase, or VHA) is a highly conserved, membrane-bound multisubunit enzyme complex composed of at least 14 different subunits, which form an activated holoenzyme of the V-ATPase, and is widely distributed in prokaryotes and eukaryotes. Functioning as an ATP-driven proton pump in various intracellular membranes, the V-ATPase regulates physiological processes of cell by controlling the acidic environment that is necessary for a variety of essential functions, especially certain organelle pathways. In eukaryotes, microfilaments, microtubules, and intermediate filaments form an inner high dynamic cytoskeleton to adapt for development of cell conformation, and sustain most of life process of cell, in which actin cytoskeleton provides inner machinery supplies. There are a close corelationm between the dynamic actin cytoskeleton and physiological functions of cells. In processes of sexual reproduction of flowering plants, pollen germination and pollen tubes growth are two key factors, in which actin cytoskeleton plays a crucial role. The actin cytoskeleton shows a precision regulation and high dynamic structure in different germinating stages and different tissues of pollen, and the dynamic assembly of actin cytoskeleton is accordingly regulated by many actin-binding proteins. Previous study showed that the V-ATPase B subunits can derictly interact with actin cytoskeleton through actin-binding sites, motifs, and domains on their N-terminals, and are involved in actin cytoskeleton dynamic remodeling, pollen germination, and pollen tube growth, and play important functions.The present study have investigated on physiological and biochemical function of Arabidopsis thaliana V-ATPase B1, B2, and B3 involved in dynamic actin cytoskeleton remodeling and of V-ATPase involved in signaling transduction of Arabidopsis thaliana growth and development.1. Analysis on characterization of V-ATPase B1, B2, and B3 genes (AtVHA-B1,-B2, and-B3) and proteins (AtVAB1, AtVAB2, and AtVAB3) with bioinformatics methods indicates that genes of AtVHA-B1 and AtVHA-B3 are located in chromosome 1, but AtVHA-B2 in chromosome 4. These genes share high identity in gene and protein sequences, but they don't share same chromosome. Though AtVHA-B1 and -B3 are located in chromosome 1, but there is a long distance between the two subunits, suggesting that these subunits may play distinct roles in cytoplasm. Additionally, there exist two potential actin-binding sites, profilin-like motif and LKXXET-like motif, in AtVAB1, AtVAB2, and AtVAB3, indicating that AtVABs play crucial function in dynamic remodeling of actin cytoskeleton and plant cell development and signaling transduction, and are involved in pollen germination, pollen tube growth, intracellular material movement, cell responding to stress, and proton transferring. Though the sequences of AtVABs share 97.27% identity, there is an unconserved region among these AtVABs, which form a potential basis of structures resulting functional differences of AtVABs, implying AtVABs play either distinct or harmonious roles during cell processing.2. Investigations on expression pattern and differences of biochemical function of AtVAB1, AtVAB2, and AtVAB3 indicate that all of AtVABs have similar expression pattern, and are involved in actin-binding,-bundling,-capping, and -severing, but they show distinct function. AtVAB1 promotes depolymerization of actin filaments, has high capping and severing activity, and inhibits F-actin elongation. AtVAB2 inhibits depolymerization of actin filaments, stabilizes F-actin, and has capping activity and lower severing activity with dose-dependent manner. But AtVAB3 regulates minutely dynamic actin cytoskeleton. In addition, activity of AtVAB1, AtVAB2, and AtVAB3 are regulated by Ca2+.3. Studies on the regulating function of AtVAB2 involving in dynamic remodeling of actin cytoskeleton in different pH and Ca2+ environments clarify that AtVAB2 functions as a actin-binding,-bundling,-capping, and -severing protein, and is involved in dynamic actin cytoskeleton and regulated significantly by pH and Ca2+ condition.4. Investigations on the roles of AtVAB2 in pollen germination and pollen tube growth, and functions of AtVAB2 in organization of actin cytoskeleton in pollen grains and pollen tubes find that loss of AtVAB2 function results more sensitive to LatB of pollen germination and pollen tube growth, unstabilizing structures of actin cytoskeleton in pollen grains and pollen tubes, decreased germination rate, and inhibited growth of pollen tubes, indicating that through stabilizing organization of actin cytoskeleton in pollen, AtVAB2 is involved in pollen germination and pollen tube growth, even cell development and cell period, and play important roles. Further analysis implies that the physiological function of AtVAB2 may be performed through two pathways. On the one hand,AtVAB2 functions as an actin-binding protein, is involved in regulation on dynamic actin cytoskeleton and affects pollen growth and development; on the other hand, AtVAB2 acts as an important subunit, regulates V-ATPase holoenzyme activity by regulating reversible assembly/disassembly of the V-ATPase, and further indirectly mediating acidification states of intracellular compartments, and affects pollen growth and development, suggesting that the V-ATPase and its subunit B may synchronously play a crucial role in cell processes5. Studies on V-ATPase involved in regulating several intracellular signaling pathways indicate that the activity of V-ATPase is regulated by various signal molecules, suggesting that V-ATPase may be required for faithful signal transduction processes regulating pollen cell, even Arabidopsis thaliana growth and survival.Taken together, in vitro and in vivo functional investigations have clarified physiological and biochemical function on AtVAB2 involving in dynamic actin cytoskeleton remodeling, indicated molecular regulating mechanism on AtVAB2 involving in plant physiological processes, which have great values to scientific theory. Additionally, the functional differences of AtVABl, AtVAB2, and AtVAB3 imply they may be involved in different physiological processes of same cell trafficking, or different regulation pathways of different physiological processes, resulting in conserved heredity of the AtVABl, AtVAB2, and AtVAB3 in long life evolution process of Arabidopsis thaliana, which will supply important theoretical basis for studying on Arabidopsis thaliana molecular genetics and development biology.
Keywords/Search Tags:Arabidopsis thaliana, V-ATPase, B subunits, Actin cytoskeleton, Dynamic assembly, Pollen germination, Pollen tube growth, Biochemical and physiological function, Signal transduction
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