Kimiharu Ishizawa

Involvement of plasma membrane H+‐ATPase in anoxic elongation of stems in pondweed (Potamogeton distinctus) turions

• 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.

Anoxia-enhanced expression of genes isolated by suppression subtractive hybridization from pondweed (Potamogeton distinctus A. Benn.) turions

Pondweed (Potamogeton distinctus A. Benn.), a monocot aquatic plant species, has turions, which are overwintering buds forming underground as an asexual reproductive organ. Turions not only survive for more than one month but also elongate under strict anoxia, maintaining high-energy charge by activation of fermentation. We cloned 82 cDNA fragments of genes, that are up-regulated during anoxic growth of pondweed turions, by suppression subtractive hybridization. The transcript levels of 44 genes were confirmed to be higher under anoxia than those in air by both Northern blot analysis and a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method. A homology search for their nucleotide sequences revealed that some of them are highly homologous to known sequences of genes from other plants. They included alcohol dehydrogenase, pyruvate decarboxylase (PDC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vacuolar H+-translocating pyrophosphatase and a plasma membrane intrinsic protein. Time courses of transcript accumulation of some genes under anoxia were different from those in air. The activity of PDC increased under anoxic conditions but the activities of GAPDH and pyrophosphatase remained constant after anoxic treatment. Anoxically up-regulated genes are possibly involved in physiological events to control energy production, pH regulation and cell growth under anoxia. These results suggest that transcriptional regulation of these genes serves as an essential part of survival and growth of pondweed turions under anoxia.

Intracellular pH Regulation of Plant Cells Under Anaerobic Conditions

The intracellular pH of living cells is strictly controlled in each compartment. Under normal conditions, the cytoplasmic pH (pHc) and the vacuolar pH (pHv) of typical plant cells are maintained at slightly alkaline (typically 7.5) and acidic (typically 5.5) values, respectively. A failure to maintain the pH homeostasis of cells leads to cell death. In general, anaerobic conditions induce acidosis in the cytoplasm of plant cells and thereby prolonged anoxia causes cell death. As a result, the regulation of intracellular pH has been an important topic for research in studies of the anoxia tolerance of plant cells (Plant Physiol 100:1–6, 1992; Annu Rev Plant Physiol Plant Mol Biol 48:223–250, 1997; Funct Plant Biol 30:1–47, 2003; Funct Plant Biol 30:999–1036, 2003; Plant Stress 2:1–19, 2008; Annu Rev Plant Biol 59:313–339, 2008). To date many researchers have published review articles to discuss acidosis and pH regulation of plant cells exposed to anaerobic conditions (Encyclopedia of plant physiology, Springer, Berlin, pp. 317–346, 1976; Annu Rev Plant Physiol 30:289–311, 1979; Int Rev Cytol 127:111–173, 1991; Ann Bot 79:39–48, 1997; Regulation of tissue pH in plants and animals, Cambridge University Press, Cambridge, pp. 193–213, 1999; Int Rev Cytol 206:1–44, 2001; Ann Bot 96:519–532, 2005; Plant roots: the hidden half, CRC Press, Boca Raton, Chapter 23, pp. 1–18, 2013). In this review, I will summarize the proposed mechanisms to control intracellular pH and include a brief discussion about anoxia tolerance on the basis of the limited information available for plant cells possessing extremely strong tolerance to anoxia.