Differential Expression of Vacuolar H+-ATPase Subunit c Genes in Tissues Active in Membrane Trafficking and Their Roles in Plant Growth as Revealed by RNAi

Abstract

Acidification of intracellular compartments by the vacuolar-type H1 -ATPases (VHA) is known to energize ion and metabolite transport, though cellular processes influenced by this activity are poorly understood. At least 26 VHA genes encode 12 subunits of the V 1 V o -ATPase complex in Arabidopsis, and how the expression, assembly, and activity of the pump are integrated into signaling networks that govern growth and adaptation are largely unknown. The role of multiple VHA-c genes encoding the 16-kD subunit of the membrane V o sector was investigated. Expression of VHA-c1, monitored by promoter-driven b-glucuronidase (GUS) activity was responsive to light or dark in an organ-specific manner. VHA-c1 expression in expanding cotyledons, hypocotyls of etiolated seedlings, and elongation zone of roots supported a role for V-ATPase in cell enlargement. Mutants reduced in VHA-c1 transcript using dsRNA-mediated interference showed reduction in root growth relative to wildtype seedlings. In contrast, VHA-c3 promoter::GUS expression was undetectable in most organs of seedlings, but strong in the root cap. Interestingly, dsRNA-mediated mutants of vha-c3 also showed reduced root length and decreased tolerance to moderate salt stress. The results suggest that V-ATPase functions in the root cap influenced root growth. Expression of VHA-c1 and VHA-c3 in tissues with active membrane flow, including root cap, vascular strands, and floral style would support a model for participation of the V o sector and V 1 V o -ATPase in membrane trafficking and fusion. Two VHA-c genes are thus differentially expressed to support growth in expanding cells and to supply increased demand for V-ATPase in cells with active exocytosis.From the perspective of physiologists and biochemists, it is well established that primary proton pumps are crucial for plant growth and survival. Among three distinct proton pumps, the most complex is the vacuolar-type H 1 -ATPase. Physiological and biochemical studies have demonstrated that acidification of the vacuolar compartment by this pump energizes the uptake and release of ions and metabolites (Sze et al., 1992;Lü ttge and Ratajczak, 1997). However, due to its structural complexity, we know very little about how the expression, assembly, and activity of this pump are integrated into the signaling networks that govern the life cycle of plants. In addition to its association with vacuolar membranes in plant cells, the pump has been localized to diverse subcellular membranes, including the Golgi (Ali and Akazawa, 1986; Matsuoka et al., 1997), endoplasmic reticulum (ER; Herman et al., 1994), intracellular vesicles, and the plasma membrane Schumacher et al., 1999;Dietz et al., 2001). Yet the specific subcellular functions of V-ATPases in distinct cell types and their consequences on a developing multicellular plant are largely unknown. Thus, from the perspectives of molecular, cell, and developmental biologists, many questions remain.Based on extensive biochemical and structural studies from animal, yeast and plant...

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

“…Previously, several mutants with reduced V-ATPase activity appeared to be hypersensitive to Na + , supporting the notion that increased V-ATPase activity is required for Na + compartmentalization under salt stress [49,50]. However, consistent with our results, the vha-a2 vha-a3 double mutant does not show hypersensitivity to external Na + , suggesting that tonoplast V-ATPase activity may not be a limiting factor for Na + sequestration and tolerance [34].…”

“…Acidification of the vacuolar compartment by the VATPase is known to energize secondary ion and metabolite transport. The cbl2 cbl3 mutant is smaller than wildtype presumably because alkalized pH in the vacuoles of cbl2 cbl3 hampers cell expansion owing to inadequate turgor pressure [49]. As well, the reduced H + transport across the tonoplast hinders the sequestration of excessive levels of cations into the vacuoles so that the cbl2 cbl3 mutant is hypersensitive to a variety of exogenous cations.…”

Roles of ion channels and transporters in guard cell signal transduction

“…At the molecular level, plant vacuolar H + -ATPases (V-ATPases), like those of other eukaryotes, have a multiple subunit composition [132]. Genes for at least 26 different V-ATPase subunits have been identified in the Arabidopsis genome, with some subunits encoded by small gene families [132].…”

“…At the molecular level, plant vacuolar H + -ATPases (V-ATPases), like those of other eukaryotes, have a multiple subunit composition [132]. Genes for at least 26 different V-ATPase subunits have been identified in the Arabidopsis genome, with some subunits encoded by small gene families [132]. By contrast, V-PPases are large homodimeric enzymes which utilize energy from pyrophosphate (PP) hydrolysis to drive H + uptake into the vacuole [133,134].…”

Peculiar features of plant cell vacuolar H+-ATPase regulation

“…К сожалению, к настоящему времени только для немно-гих из изоформ растительной V-АТФазы проведен анализ изоформ-специфичной экспрессии. Например, показано, что экспрессия гена VHA-c1 происходит в зоне растяжения корня арабидопсиса и кончика корня, в отличие от VHA-c3, экспресси-руемого в клетках корневого чехлика и пыльце [16]. Сходные данные были получе-ны на растениях хлопка, где накопление изоформ VHA-c было идентифицировано в быстро и интенсивно растягивающихся клетках [17].…”