Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2',7'-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and 31P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by 31P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested.

No abstract

We studied physiological roles of the yeast vacuole in the phosphate metabolism using 31P-in vivo nuclear magnetic resonance (NMR) spectroscopy. Under phosphate starvation wild-type yeast cells continued to grow for two to three generations, implying that wild-type cells contain large phosphate pool to sustain the growth. During the first four hours under the phosphate starved condition, the cytosolic phosphate level was maintained almost constant, while the vacuolar pool of phosphate decreased significantly. 31P-NMR spectroscopy on the intact cells and perchloric acid (PCA) extracts showed that drastic decrease of polyphosphate took place during this phase. In contrast, delta slp1 cells, which were defective in the vacuolar compartment, thus lacked polyphosphate, ceased their growth immediately when they faced to phosphate starvation. Taken together, we conclude that vacuolar polyphosphate provides an active pool for phosphate and is mobilized to cytosol during phosphate starvation and sustained cell growth for a couple rounds of cell cycle.

N-Acetylchitoheptaose strongly induces a set of defense reactions in suspension-cultured rice cells including cytoplasmic acidification (K. Kuchitsu, Y. Yazaki, K. Sakano, and N. Shibuya, Plant Cell Physiol. 38:1012-1018, 1997) and the accumulation of mRNAs for two rapidly activated genes, EL2 and EL3 (E. Minami, K. Kuchitsu, D.-Y. He, H. Kouchi, N. Midoh, Y. Ohtsuki, and N. Shibuya, Plant Cell Physiol. 37:563-567, 1996), as well as phenylalanine ammonia-lyase (PAL), chitinase and β-1,3-glucanase. Treatment of cells with propionic acid resulted in the accumulation of the mRNAs for EL2, EL3, and PAL in a manner similar to the accumulation induced by N-acetylchitoheptaose. Concomitantly, there was a rapid decrease in the cytoplasmic pH as detected with in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Interestingly, K-252a, a potent inhibitor of Ser/Thr protein kinases, strongly inhibited gene induction by N-acetylchitoheptaose, but showed much less inhibition of gene induction caused by propionic acid. Calyculin A, a protein phosphatase inhibitor, induced mRNA accumulations for EL2, EL3 and PAL, with concomitant acidification of the cytoplasm. On the other hand, chitinase and β-glucanase mRNA levels did not change after addition of propionic acid or calyculin A. Treatment of the cells with propionic acid did not induce the production of reactive oxygen species. These results strongly suggest that cytoplasmic acidification at the early stage of elicitor action could be a key step in the signal transduction events leading to the expression of elicitor-responsive genes. A hypothetical model of elicitor signal pathway is proposed based on these results.

The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.

Summary• Pondweed (Potamogeton distinctus) turions can elongate in the absence of O 2 . Alcoholic fermentation serves to produce energy for anoxic elongation via the breakdown of starch stored in cells. However, the mechanism of cell growth during anoxic elongation is not fully understood.• Changes in pH, H + equivalent and lactate content of the incubation medium were measured during anoxic elongation. The effects of fusicoccin (FC), indole-3-acetic acid (IAA), vanadate, erythrosine B and K + channel blockers on anoxic elongation were examined. Cytoplasmic pH and vacuolar pH were measured by 31 P nuclear magnetic resonance (NMR) spectroscopy.• Acidification of the incubation medium occurred during anoxic elongation. The contribution of CO 2 and lactic acid was not sufficient to explain the acidification. FC and IAA enhanced the elongation of stem segments. Vanadate and erythrosine B inhibited anoxic elongation. Acid growth of notched segments was observed. The activity of plasma membrane H + -ATPase extracted from pondweed turions was increased slightly in anoxic conditions, but that from pea epicotyls sensitive to anoxic conditions was decreased by incubation in anoxic conditions. Both the cytoplasmic pH and vacuolar pH of pondweed turion cells chased by 32 P NMR spectroscopy were stabilized during a short period < 3 h after anoxic conditions.• We propose that the enhancement of H + extrusion by anoxic conditions induces acidification in the apoplast and may contribute to the stabilization of pH in the cytoplasm.

No abstract