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.     

Stimulation of glycolysis in anaerobic elongation of pondweed (Potamogeton distinctus) turions

Stem segments prepared from pondweed (Potamo geton distinctus A. Benn.) turions (overwintering buds) elongate in anaerobic conditions, whereas there is almost no elongation in air. The anaerobic elongation was accompanied by a decrease in dry weights of stem segments, mainly due to consumption of storage starch in the amyloplasts of stem cells. On the other hand, total contents of amino acids increased in stem segments, in which contents of alanine, valine, leucine, and isoleucine increased, but contents of asparatic acid decreased. Moreover, contents of lactate in stem tissues increased at an early stage of anaerobic incubation. In tracer experiments with 14C-glucose, 14C incorporation into stem tissues in anoxia was only half of that in normoxia. However, conversion of 14C to ethanol occurred exclusively in anoxia. 14C-labelled metabolites were analysed by two-dimensional cellulose thin-layer chromatography. 14C incorporation into sucrose and alanine was significantly increased in anoxia. The activity of alanine aminotransferase was enhanced by anoxia, suggesting that pyruvate is a precursor of alanine synthesis. The results suggest that pondweed turions produce energy necessary for anaerobic elongation by activating conversion of storage starch in the amyloplasts to ethanol, alanine and lactate.     

Growth and energy status of arrowhead tubers,pondweed turions and rice seedlings under anoxic conditions

The responses of two aquatic plants, arrowhead (Sagittaria pygmaea Miq.) and pondweed (Potamogeton distinctus A. Benn), to anoxia were compared with those of rice (Oryza sativa L.). Shoot elongation of arrowhead tubers was enhanced at around 1 kPa O₂, whereas that of pondweed turions was slight in air and reached a maximum in the absence of O₂. Anaerobic enhancement of alcohol dehydrogenase (ADH) activity took place in rice coleoptiles but not in arrowhead and pondweed shoots. Shoots of both arrowhead and pondweed maintained a more stable energy status than did the rice coleoptile under anaerobic conditions. Total adenylate nucleotide contents of arrowhead and pondweed shoots were constant under anaerobic conditions. Adenylate energy charge in both shoots remained at a high and stable level of more than 0·8 for at least 8 d. Three forms of ADH from arrowhead shoots were separated by starch gel electrophoresis, showing that the activity of each ADH form was different under aerobic and anaerobic conditions. The incorporation of ³⁵S-labelled Cys and Met into soluble proteins in arrowhead shoots showed active protein biosynthesis and an involvement of a special set of polypeptides in the anaerobiosis.     

Evidence for the Critical Role of Sucrose Synthase for Anoxic Tolerance of Maize Roots using a Double Mutant

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O₂, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O₂. In this situation, SuSy plays a critical role in anoxic tolerance.     

Sucrose Utilization via Invertase and Sucrose Synthase with Respect to Accumulation of Cellulose and Callose Synthesis in Wheat Roots under Oxygen Deficiency

The sucrose cleavage by sucrose synthase (SuSy) and neutral invertase was studied in wheat roots (Triticum aestivum L.) subjected to hypoxia or anoxia for 4 days. By in situ activity staining, increased SuSy activity was observed in the tip region and stele of root axes while the activity of invertase decreased. Cellulose content significantly increased in hypoxically treated roots. The cellulose deposition was correlated with regions of high SuSy activity, being mainly located in the pericycle and endodermis. Invertase activity was distributed along the root without clear difference between cortex and stele. Under root hypoxia, a significant increase in the structural carbohydrates, callose and especially cellulose, was shown. Increasing levels of soluble carbohydrates were partially used to synthesize cellulose for secondary wall thickening and callose to counteract the tissue injury following low-oxygen stress. Under strict anoxia, the roots were much more injured but sustained a high level of cellulose and callose while the soluble carbohydrates almost disappeared.     

Comparative analysis of anoxic coleoptile elongation in rice varieties: relationship between coleoptile length and carbohydrate levels, fermentative metabolism and anaerobic gene expression

Rice ( Oryza sativa L.) seeds can germinate under anoxia and can show coleoptile elongation. The anoxic coleoptile is usually longer than aerobic coleoptiles. Although several hypotheses have been proposed to explain the ability of rice to elongate coleoptiles under anoxia, conclusive experimental evidence explaining this physiological trait is lacking. In order to investigate whether metabolic and molecular markers correlate with anoxic coleoptile length, we screened 141 Italian and 23 Sri Lankan rice cultivars for their ability to elongate coleoptiles under anoxia. Differences in anoxic coleoptile length were used to evaluate whether a correlation exists between coleoptile length and biochemical and molecular parameters. The expression of genes coding for glycolytic and fermentative enzymes showed a very low correlation with anoxic coleoptile length. Although differences were found in carbohydrate content between the varieties tested, this parameter also does not appear to be critical in terms of coleoptile elongation. Efficient ethanol fermentation does, however, correlate well with the elongation of coleoptiles under anoxic conditions.     

Anoxia tolerance and α-amylase activity in four rice cultivars

The relationship between anoxia tolerance and reserved carbohydrate catabolism was investigated in four rice (Oryza sativa L.) cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress, however, the elongation of cvs Koshihikari and Awa-akamai was much greater than that of cvs Touzoumochi and Asahimochi. The anoxic coleoptiles of cvs Koshihikari and Awa-akamai contained about 2-fold as much ATP as those of cvs Touzoumochi and Asahimochi. Ethanol production in the anoxic coleoptiles of cvs Koshihikari and Awa-akamai was about 2-fold as much as that of cvs Touzoumochi and Asahimochi, which suggests that ethanolic fermentation is probably more active in cvs Koshihikari and Awa-akamai than in cvs Asahimochi and Touzoumochi. Activity of α-amylase, which catabolizes starch to soluble sugars, in endosperms of cvs Koshihikari and Awa-akamai was about 2-fold that of cvs Touzoumochi and Asahimochi, and soluble sugar concentration in the coleoptiles of cvs Koshihikari and Awa-akamai was about 3-fold greater than that of cvs Touzoumochi and Asahimochi. Soluble sugar concentration and ethanol production rate in the coleoptiles of rice seedlings were correlated well with α-amylase activity in their endosperms, which were also correlated well with anoxia tolerance with respect to the coleoptile elongation and ATP concentration in the coleoptiles. These results suggest that the ability to degrade starch to soluble sugar by α-amylase in endosperm may be important for the anoxia tolerance in rice coleoptiles and it may serve to distinguish the anoxia tolerance of rice coleoptiles.     

Physiological and metabolic adaptations of Potamogeton pectinatus L. tubers support rapid elongation of stem tissue in the absence of oxygen

Tubers of Potamogeton pectinatus L., an aquatic pondweed, over-winter in the anoxic sediments of rivers, lakes and marshes. Growth of the pre-formed shoot that emerges from the tuber is remarkably tolerant to anoxia, with elongation of the stem occurring faster when oxygen is absent. This response, which allows the shoot to reach oxygenated waters, occurs despite a 69-81% reduction in the rate of ATP production, and it is underpinned by several physiological and metabolic adaptations that contribute to efficient energy usage. First, extension of the pre-formed shoot is the result of cell expansion, without the accumulation of new cellular material. Secondly, after over-wintering, the tuber and pre-formed shoot have the enzymes necessary for a rapid fermentative response at the onset of growth under anoxia. Thirdly, the incorporation of [(35)S]methionine into protein is greatly reduced under anoxia. The majority of the anoxically synthesized proteins differ from those in aerobically grown tissue, implying an extensive redirection of protein synthesis under anoxia. Finally, anoxia-induced cytoplasmic acidosis is prevented to an unprecedented degree. The adaptations of this anoxia-tolerant plant tissue emphasize the importance of the mechanisms that balance ATP production and consumption in the absence of oxygen.     

Sucrose accumulation in sweet sorghum stem internodes in relation to growth

Sweet sorghum (Sorghum bicolor L. Moench) stems of different cultivars (NK 405. Keller and Tracy) reveal a different pattern of sucrose accumulation with respect to in-ternodal sugar content and distribution. The onset of sucrose storage is not necessarily associated with the reproductive stage of the plant, as was hitherto assumed, but obviously occurs after cessation of internodai elongation as was postulated for the sugarcane stem. For at least two of the three cultivars, ripening is an internode to internode process beginning at the lowermost culm parts. Intensive growth of the internodes, combined with a high hexose content in stern parenchyma, shows a strong positive correlation (r |Mg 0.94) to the activity of sucrose synthase (SuSy; EC 2.4.13), but not to invertase (EC 3.2.1.26) which is not present as soluble (neutral and acid) or cell wall-bound, salt-extractable enzyme in the three culsivars investigated. Sucrose synthase measured in sucrose cleavage and synthesis direction reveals divergent activity rates and sensitivity towards exogenously applied Mg²⁺ ions and pH. SuSy activity is connected to the increase of internodai sucrose content in so far as (1) its decline is a prerequisite for the onset of sucrose accumulation and (2) it remains at a constant low level during sucrose storage. Sucrose phosphate synthase (SPS; EC 2.4.1.14) activity in the sorghum stem is low compared to SuSy and uniformly distributed over all inter-nodes. Only source leaves of sorghum show a considerable SPS activity, but neither stem nor leaf SPS reveal a positive correlation to the increase of internodai sucrose content. Sucrose phosphate phosphatase (SPP; EC 3.1.3.24) amounts lo only 24–30% of the respective SPS activity but follows the same distribution pattern. None of the enzymes under study proves to be responsible for the extent of sucrose storage in the stem, so other phenomena such as transport processes within the stern tissue require further investigation.     

Carbohydrate metabolism in germinating caryopses of <Emphasis Type="Italic">Oryza sativa</Emphasis> L. exposed to prolonged anoxia

Anoxia tolerance can be evaluated not only in terms of growth or survival of plant organs during oxygen deprivation, but also in relation to carbohydrate utilization in the context of a well-modulated fermentative metabolism. Rice (Oryza spp.) is unique among cereals, in that it has the distinctive ability to germinate under complete anaerobiosis by using the starchy reserves in its seeds to fuel the anaerobic metabolism. The aim of the present study was to evaluate the ability of germinating rice seedlings to survive a long-term oxygen deficiency [40 days after sowing (DAS)] and the effects on sugar metabolism, focusing on starch degradation as well as soluble sugars transport and storage under anoxia. No significant decline in vitality occurred until 30 DAS though no recovery was detected following longer anoxic treatments. Growth arrest was observed following anoxic treatments longer that 20 DAS, in concomitance with considerably lower ethanol production. Amylolytic activity in embryos and endosperms had similar responses to anoxia, reaching maximum content 30 days after the onset of stress, following which the levels declined for the remainder of the experiment. Under anoxia, average amylolytic activity was twofold higher in embryos than endosperms. Efficient starch degradation was observed in rice under anoxia at the onset of the treatment but it decreased over time and did not lead to a complete depletion. Our analysis of α-amylase activity did not support the hypothesis that starch degradation plays a critical role in explaining differences in vitality and coleoptile growth under prolonged oxygen deprivation.     

Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus

Potamogeton crispus is a cosmopolitan aquatic species and is widely used as a pioneer species for vegetation restoration of eutrophic lakes. However, many restoration projects applying P. crispus turions have not been successful. Earlier studies focused on effects of light and temperature on turion germination. The purpose of this study was to determine whether sediment anoxia and light interactively affected the turion germination and early growth of P. crispus. Anoxic conditions in the experiment were produced by adding sucrose to the sediment. The germination rate of the turions was 68–73% lower in the highly anoxic condition treatment than in the control. Medium light intensity (10% of natural light at the water surface) was more favorable for germination under slightly anoxic conditions than either low or high light intensity. The growth of newly-formed sprouts was also significantly inhibited by sediment anoxia. Photosynthesis and shoot biomass were reduced under sediment anoxia, whereas total chlorophyll content, root biomass, and soluble protein content were highest in the low anoxic condition treatment. Medium light improved net photosynthesis and biomass production of the sprouts. We conclude that turion germination and sprout growth can be significantly inhibited by sediment anoxia. Medium light intensity may alleviate this inhibition by anoxia, but light has little effect when sediment anoxia is severe. For the purposes of vegetation restoration, more attention should be paid to the role of sediment anoxia, and it is necessary to improve sediment and light conditions for turion germination and early growth of P. crispus in eutrophic lakes. These results will contribute to a more complete understanding of turion germination dynamics of P. crispus and will be useful for future restoration programs. Handling editor: S. M. Thomaz     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.