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(3,11,19,22,31,32) while the Ca2+-ATPases are associated with the ER and plasma membrane (6,7,9,11,17,28,31). It has been suggested that both types oftransporter may exist in a single membrane type (2, 30) although this has not been investigated in higher plants. We examined the Ca2+-transport activity of microsomes isolated from barley roots in order to determine which ofthe two types ofCa2+ transporters were associated with the major organelle fractions.The mechanisms by which Ca2+ is transported out of the cytoplasm against a large concentration gradient can be studied using isolated membrane vesicles (3,6,7,11,17,22,28,31,32 (13)(14)(15)(16). In the present paper the mechanisms and capacity for formation of Ca2`gradients by these vesicles are assessed. A prominent Ca2+/nH+ antiporter activity in tonoplast vesicles is characterized and evidence for a non-ER, non-plasma membrane Ca2+-ATPase is presented. A distinction is made between the activity of the Ca24/nH' antiporter and independent effects of Ca2+ on the formation and maintenance of ApH by the PM-and V-ATPases.Precise regulation ofcytoplasmic Ca2" may be an important element of signal transduction in plant cells (23,27), as it is in animal cells (12). Resting levels of cytoplasmic Ca2" are reported to be less than 0.5 AM in plants (8,18,26). These low levels ofcytoplasmic Ca2" are maintained by active uptake into organelles and export across the plasma membrane (7,12,26). To date, two types of Ca2" transport activity have been described in plant cells, one which is dependent on the MATERIALS AND METHODS Plant MaterialSeeds of barley (Hordeum vulgare L. cv CM72) were sown above an aerated solution containing full strength nutrients 2 Abbreviations: ApH, difference in pH across the vesicle membrane; ESR, electron spin resonance; PM-ATPase, plasma membrane H+-ATPase; PM, plasma membrane-enriched membrane fraction; 6,"5N,2,6,6, 1-'5N-1-oxyl; TN, tonoplast-enriched membrane fraction; Q, fluorescence quench; V-ATPase, vacuolar-type H+-ATPase; DCCD, dicyclohexylcarbodiimide; nH, number of H+ where n is unknown; BTP, bistris propane.
(3,11,19,22,31,32) while the Ca2+-ATPases are associated with the ER and plasma membrane (6,7,9,11,17,28,31). It has been suggested that both types oftransporter may exist in a single membrane type (2, 30) although this has not been investigated in higher plants. We examined the Ca2+-transport activity of microsomes isolated from barley roots in order to determine which ofthe two types ofCa2+ transporters were associated with the major organelle fractions.The mechanisms by which Ca2+ is transported out of the cytoplasm against a large concentration gradient can be studied using isolated membrane vesicles (3,6,7,11,17,22,28,31,32 (13)(14)(15)(16). In the present paper the mechanisms and capacity for formation of Ca2`gradients by these vesicles are assessed. A prominent Ca2+/nH+ antiporter activity in tonoplast vesicles is characterized and evidence for a non-ER, non-plasma membrane Ca2+-ATPase is presented. A distinction is made between the activity of the Ca24/nH' antiporter and independent effects of Ca2+ on the formation and maintenance of ApH by the PM-and V-ATPases.Precise regulation ofcytoplasmic Ca2" may be an important element of signal transduction in plant cells (23,27), as it is in animal cells (12). Resting levels of cytoplasmic Ca2" are reported to be less than 0.5 AM in plants (8,18,26). These low levels ofcytoplasmic Ca2" are maintained by active uptake into organelles and export across the plasma membrane (7,12,26). To date, two types of Ca2" transport activity have been described in plant cells, one which is dependent on the MATERIALS AND METHODS Plant MaterialSeeds of barley (Hordeum vulgare L. cv CM72) were sown above an aerated solution containing full strength nutrients 2 Abbreviations: ApH, difference in pH across the vesicle membrane; ESR, electron spin resonance; PM-ATPase, plasma membrane H+-ATPase; PM, plasma membrane-enriched membrane fraction; 6,"5N,2,6,6, 1-'5N-1-oxyl; TN, tonoplast-enriched membrane fraction; Q, fluorescence quench; V-ATPase, vacuolar-type H+-ATPase; DCCD, dicyclohexylcarbodiimide; nH, number of H+ where n is unknown; BTP, bistris propane.
One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots takes place when the plant plasma membrane (PM) invaginates around the fungal arbuscular structures resulting in the periarbuscular membrane formation. To investigate whether AM symbiosis-specific proteins accumulate at this stage, two complementary MS approaches targeting the root PM from the model legume Medicago truncatula were designed. Membrane extracts were first enriched in PM using a discontinuous sucrose gradient method. The resulting PM fractions were further analysed with (i) an automated 2-D LC-MS/MS using a strong cation exchange and RP chromatography, and (ii) SDS-PAGE combined with a systematic LC-MS/MS analysis. Seventy-eight proteins, including hydrophobic ones, were reproducibly identified in the PM fraction from non-inoculated roots, representing the first survey of the M. truncatula root PM proteome. Comparison between non-inoculated and Glomus intraradices-inoculated roots revealed two proteins that differed in the mycorrhizal root PM fraction. They corresponded to an H(+)-ATPase (Mtha1) and a predicted glycosylphosphatidylinositol-anchored blue copper-binding protein (MtBcp1), both potentially located on the periarbuscular membrane. The exact role of MtBcp1 in AM symbiosis remains to be investigated.
Abstract.The primary or secondary energized transport of Ca 2+, Mg 2 § and H § into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca 2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca 2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Cae+-pumping and tonoplast H+-pyrophosphatase (PP~ase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca2 § activity could be detected. The ATP-dependent Ca 2 § uptake into tonoplast vesicles (using Ca 2+ concentrations from 0.8-1 gM) was completely inhibited by the Ca 2 § ionophore ionomycin (1 gM) whereas the protonophore nigericin (1 gM) which eliminates ATPdependent intravesicular H § accumulation had no effect. Vanadate (IC5o = 43 gM) and diethylstilbesterol (IC50 =5.2 laM) were potent inhibitors of this type of Ca 2+ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%-50% of the ATP-dependent activity (K m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca 2 § transport mechanism is the only functioning Ca 2 § transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca 2+ concentration. In contrast, the secondary energized Ca2+-transport mechanism of the tonoplast, the low-affinity Ca 2 § § which was reported to allow the uptake of Ca 2+ in exchange for H § functions chiefly as an Mg 2 § transporter under physiological conditions because cytosolic Mg 2+ is several orders of magnitude higher than the Ca 2+ concentration. This conclusion was deduced from experiments showing that Mg 2 § ions in a concentration range of 0.01 to 1 mM Abbreviations: DES=diethylstilbesterol; DCCD=N,N'-dicyclohexylcarbodiimide; CCR = cytochrome-c reductase; IC5o = concentration which causes 50% inhibition; IDPase=inosine diphosphatase; Indo 1-1-(2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2 '-amino-5' methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid; PP~ase = tonoplast H +-pyrophosphatase Correspondence to: A. Hager; FAX: 49(7071) 293287 triggered a fast efflux of H-from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H+-ATPase and H+-PPiase was found to be influenced by Ca 2 § differently from and independently of the Mg 2+ concentration. Calcium was a strong inhibitor for the H § (IC50 = 18 ~tM, Hill coefficient nH=l.7) but a weak one for the H+-ATPase (IC50 = 330 gM, nH= 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca 2 § and Mg2+-regulated H § PPiase, (ii) the newly described high-affinity CaZ+-ATPase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the H §
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