Fatty acids of rice coleoptiles in air and anoxia

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [¹⁴C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O₂, as it does in other plants.     

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.     

Exogenous Nitrate as a Terminal Acceptor of Electrons in Rice (Oryza sativa) Coleoptiles and Wheat (Triticum aestivum) Roots under Strict Anoxia

To elucidate the physiological role of exogenous nitrate under anaerobic conditions, we studied the effect of 10 mM KNO₃ on the mitochondrial ultrastructure in rice (Oryza sativa L.) coleoptiles and in wheat (Triticum aestivum L.) roots, detached from four-day-old seedlings, under strict anoxia. In wheat roots, following 6-h-long anoxia in the absence of exogenous nitrate, the mitochondrial membranes were partially degraded and, after 9 h under anoxia, the mitochondrial membranes and the membranes of other organelles were completely destroyed. In rice coleoptiles, the partial membrane degradation was observed only after 24 h and their complete breakdown after 48 h of anaerobiosis. In the presence of exogenous nitrate, no membrane destruction was noticed even after 9 and 48 h of anaerobiosis in wheat roots and rice coleoptiles, respectively. These results indicate that exogenous nitrate exerts protective action as a terminal electron acceptor, alternative to the molecular oxygen. Our findings are compared with the results of other researchers concerning the adverse or favorable nitrate action on plant growth, metabolism, and energy status under anaerobic stress.     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of ¹⁴CO₂ from tissues fed with [¹⁴C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.     

Effects of ethanol on growth of rice seedlings

Effects of ethanol, the end product of ethanolic fermentation, on the growth of rice (Oryza sativa L.) seedlings were determined as a means of evaluating growth responses under anoxia. The ethanol concentrations in roots and coleoptiles of the seedlings subjected to 48 h-anoxia, and in their culture medium were 23 and 32 µmol g^–1 fresh weight, and 19 µmol ml^–1, respectively. The growth of the roots and coleoptiles of the seedlings was restricted by exogenous ethanol at concentrations above 50 mM and 100 mM, respectively, suggesting that the roots are more sensitive to ethanol than the coleoptiles.     

Anoxia tolerance and ethanol sensitivity of rice and oat coleoptiles

Anoxia tolerance and ethanol sensitivity of rice (Oryza sativa L.) and oat (Avena sativa L.) seedlings were evaluated to clarify their growth habit in anoxia. Anoxic stress inhibited elongation and dry weight gain of coleoptiles of the oat and rice seedlings; however, the inhibition of the oat coleoptiles was much greater than that of the rice coleoptiles. Anoxic stress increased endogenous ethanol concentration and alcohol dehydrogenase activity in oat and rice coleoptiles and their increases in the rice coleoptiles were much greater than those in the oat coleoptiles. At concentrations greater than 30 mM and 300 mM, exogenously applied ethanol inhibited the elongation and weight gain for the oat and the rice coleoptiles, respectively, and the inhibition was increased with increasing ethanol concentrations with marked inhibition being achieved on the oat coleoptiles. These results suggest that anoxia tolerance and induction of ethanolic fermentation in anoxia may be greater in rice than oat, and ethanol sensitivity of rice may be lower than that of oat.     

The root form of ferredoxin-NADP oxidoreductase is expressed in rice coleoptiles for the assimilation of nitrate

Two ferredoxin-dependent proteins, nitrite reductase and glutamate synthase, play a role in nitrate assimilation during the anaerobic germination of rice (Oryza sativa L.). This paper reports the expression of the root form of ferredoxin-NADP⁺ oxidoreductase (FNR), the protein responsible for providing reduced ferredoxin in rice coleoptiles. Using an antibody against FNR, a protein with the expected molecular mass for root FNR (35 kDa) was recognized by Western blot analysis in extracts from aerobic and anaerobic coleoptiles. The enzyme is synthesized de novo, as shown by immunoprecipitation of the radiolabeled 35-kDa protein from anaerobic seedlings grown in the presence of [³⁵S]methionine. Northern blot analysis with specific probes for root and leaf FNR showed the presence of mRNA for the root form but not for the leaf form, in both aerobic and anaerobic rice coleoptiles. The inductive effect of exogenous nitrate on the expression of FNR is further evidence for the presence of the root type of FNR in rice coleoptiles. The importance of the expression of root FNR during the anaerobic development of rice seedlings is discussed. Received: 7 October 1996 / Accepted: 22 January 1997     

Effects of anoxia on mitochondrial biogenesis in rice shoots: modification of in organello translation characteristics

Shoots of germinating rice (Oryza sativa L.) seedlings are able to grow under anoxia and to withstand long periods of anoxic treatment. Mitochondria were purified from aerobically germinated and anaerobically treated rice shoots by differential and isopycnic centrifugation and were found to consist of two subpopulations. The mitochondrial subpopulation of higher density was used for further characterization. Ultrastructural studies showed anaerobic mitochondria to be significantly different from aerobic mitochondria, with a matrix of lower density and more developed cristae. Aerobic and anaerobic mitochondria also differed in their specific activities for fumarase and succinate dehydrogenase, which were significantly lower after the anoxic treatment. In vivo labeling of seedlings with l-[³⁵S]methionine and subsequent isolation of the mitochondria indicated that anoxia induced a drastic decrease, but not a total inactivation, of the synthesis of mitochondrial proteins. In organello protein synthesis showed that anaerobic mitochondria were able to synthesize most of the polypeptides synthesized by aerobic mitochondria, although only in the presence of exogenous ATP, as would occur under anoxia. Anaerobic mitochondria, but not aerobic mitochondria, could carry out protein synthesis without a functional respiratory chain. Thus, mitochondrial protein synthesis was found to be potentially functional in the rice shoot under anoxia.     

Membrane-associated binding sites for indoleacetic acid in the rice coleoptile

As described previously, the sensitivity of rice (Oryza sativa L.) coleoptiles to auxin is modulated by oxygen. Under anoxia, coleoptile elongation is insensitive to exogenously applied indole-3-acetic acid (IAA), whereas its sensitivity increases in air in the presence of the exogenous stimulus. Here we report the presence of two independent classes of membrane-bound IAA-binding sites in air-grown coleoptiles. Their binding activity is strictly correlated with the system's sensitivity to IAA. We designate them as site A (high affinity) and site B (low affinity). Site A shows a relatively fast response to anoxia, and is highly specific for auxins. Regulation of site-A binding activity through ATP, whose availability decreases under anoxia, is postulated. A role as auxin carrier is suggested for site B.Abbreviations ABS(s) auxin-binding site(s) - IAA indole-3-acctic acid - NAA 2-naphthaleneacetic acid - ION3 valinomycin, nigericin, carbonylcyanide p-trifluoromethoxyphenyl hydrazone Dedicated to the memory of Professor G. Torti, who passed away on 2 May, 1988     

Pyruvate metabolism in rice coleoptiles under anaerobiosis

Relative importance of ethanolic, lactate and alanine fermentation pathways was estimated in coleoptiles of rice seedlings (Oryza sativa L.) subjected to anoxic stress. The in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and alanine aminotransferase (AlaAT, EC 2.6.1.2) in the coleoptiles increased in anoxia, whereas no significant increase was measured in lactate dehydrogenase (LDH, EC 1.1.1.27) activity. At 48 h, the ADH, PDC and AlaAT activities in anoxic coleoptiles were 62-, 15- and 7.6-fold greater, respectively, than those in the presence of oxygen. Ethanol and alanine in the coleoptiles accumulated rapidly under anoxia, increasing by 48 h, 57- and 5.6-fold compared with those in the presence of oxygen, respectively. However, lactate concentration did not increase and no initial burst of lactate production was detected. The relative ratio of carbon flux from pyruvate to ethanol, lactate and alanine in anoxic coleoptiles was estimated to be 92, 1 and 7% of the total carbon flux, respectively. These results suggest that the potential carbon flux from pyruvate to ethanol may be much greater than the potential flux from pyruvate to lactate and alanine in rice coleoptiles during anoxia.     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Effects of submergence on development and gravitropism in the coleoptile of Oryza sativa L.

Caryopses of rice (Oryza sativa L. cv. Sasanishiki) were germinated in air or under water. In submerged seedlings a twofold increase in coleoptile growth rate and an inhibition of root growth was observed. The amount of starch in the amyloplasts of submerged coleoptiles was substantially reduced compared to the air-grown control plants and plastids had a proplastidic character. During the rapid elongation of coleoptiles under water, the osmotic concentration of the press sap remained constant, whereas in air-grown coleoptiles a decrease was measured. Determination of curvature of gravistimulated air-grown and submerged shoots was carried out by placing the coleoptiles horizontally in air of 98% relative humidity. Air-grown coleoptiles reached a vertical orientation within 5 h after onset of gravistimulation. In coleoptiles germinated under water the first signs of consistent negative gravitropic bending occurred after 4–5 h and curvature was complete after 24 h. During the first 5 h of gravistimulation the water-grown coleoptiles grew at an average rate of 0.39 mm·h^–1, whereas in air-grown coleoptiles a rate of 0.27 mm·h^–1 was measured. Concomitant with the delayed onset of gravitropic bending of the water-grown coleoptiles, a change in plastid ultrastructure and an increase in starch content was observed. We conclude that the gravitropic responsiveness of the rice coleoptile depends on the presence of starch-filled amyloplasts.We wish to thank H.-J. Ensikat for technical assistance with the scanning electron microscopy. Supported by the Bundesminister für Forschung und Technologie and the Deutsche Forschungsgemeinschaft.     

Ethanol sensitivity of rice and oat coleoptiles

The ability to avoid the ethanol-induced injury was evaluated in rice ( Oryza sativa L.) and oat ( Avena sativa L.) coleoptiles. The growth of the rice and oat coleoptiles was inhibited by ethanol exogenously applied at concentrations greater than 200 and 30 m M , respectively. At 300 m M ethanol, oat coleoptiles were brown and flaccid but rice coleoptiles did not show any visible symptoms of toxicity. The acetaldehyde level in rice and oat coleoptiles was increased by exogenously applied ethanol and the increases were greater in oat than in rice coleoptiles under aerobic and anaerobic conditions. At 300 m M ethanol, the acetaldehyde concentrations in the rice and oat coleoptiles were 46 and 87 nmol g⁻¹ FW under aerobic conditions, respectively, and 52 and 124 nmol g⁻¹ FW under anaerobic conditions, respectively. The activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) in the direction of ethanol to acetaldehyde was greater in oat than in rice coleoptiles and ADH protein in oat coleoptiles was more induced by exogenously applied ethanol than that in rice coleoptiles. These results suggest that in vivo conversion rate of ethanol to acetaldehyde by ADH is lower in rice than oat coleoptiles, which may be one of the reasons that ethanol sensitivity of rice is much lower than that of oat coleoptiles. The great ability of rice to avoid the ethanol-induced injuries may contribute its anoxia tolerance when glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of energy under anaerobic conditions.     

The Effect of Helminthosporic Root Rot on the Composition of Acyl Lipids of Wheat Seedlings

The changes of the molecular species composition of esterified fatty acids (FAs) of total and nonextractable lipids were determined in roots and etiolated shoots of 3- to 10-day-old wheat (Triticum aestivumL.) seedlings infected with the fungus Bipolaris sorokiniana, the agent of helminthosporic root rot. A novel technique of assessing the extent of the infection-induced deviation of FA composition, mol %, from the control value was developed. It consists in the quantitative determination of both the deviations in this composition and the extent of contribution of separate FA species to the deviations observed. The application of this technique has shown that, for the total lipids, the maximum of such a deviation, in accordance with the membrane theory of stress, directly coincided in time with the onset of a decrease in the dry matter content in both roots and shoots. In each of these, the deviation was primarily caused by the change in the content of those FA species that usually dominate in a specific group of membrane lipids prevailing in a given organ, viz., plastid glycolipids in shoots and extraplastidal membrane phospholipids in roots. In both cases, C₂₀–C₂₂FAs significantly contributed to the deviations observed. This fact seems to reflect an enhanced formation of epicuticular waxes rich in these FAs on the shoot and root surfaces as an adaptive response of plants to fungal infection. Nonextractable (annular) membrane lipids, because of their vital importance for the survival of plant cells, differed from the total lipids with a far greater stability of their quantitative FA composition under conditions of infection-induced metabolic disturbances.     

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.     

Environmental Anoxia is Unnecessary for Inhibiting Chloroplast Photomorphogenesis in Rice Coleoptiles (Oryza sativa L.)

High population densities of germinating rice seedlings in initiallyair-saturated sealed aquatic environments exhibited d^–seedling growth consisting solely of coleoptile emergence inlight and dark environments. Residual oxygen tensions of 17–23%of the initially air-saturated water containing the d^–seedlings were evident after 15 d in both the light and dark.Coleoptiles of all d^– seedlings were stark white in appearance,lacked protochlorophyllide, and contained proplastids and amyloplasts,there being no evidence of normal etioplast development in thelight or dark and no chloroplast development in the light. Thus,complete environmental anoxia was observed to be unnecessaryfor inhibiting normal chloroplast photomorphogenesis in coleoptilesof light-germinated rice seedlings. Increasing the oxygen tensionsof the 15-d-old aquatic environments of light- and dark-germinatedd^– seedlings placed in the light resulted in normal chloroplastphotomorphogenesis in coleoptiles, shoots, and roots. Key words: Oryza sativa, environmental anoxia, chloroplast photomorphogenesis, rice coleoptiles     

Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice

Vacuolar H⁺-pyrophosphatase (V-PPase) expression increases in a number of abiotic stresses and is thought to play a role in adaptation to abiotic stresses. This paper reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using flood tolerant and intolerant cultivars to test our hypothesis. Quantitative PCR analysis showed that one vacuolar H⁺-pyrophosphatase (OVP3) was induced by anoxia, particularly in flood-tolerant rice. Regulation of OVP3 expression under anoxia was investigated by analysing putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the putative promoters of the other five OVP genes. GUS activity in transgenic rice plants containing the OVP3 promoter region linked to the GUS reporter gene was induced only by anoxia. Salt and cold treatments had little effect on OVP3 promoter-driven GUS expression when compared to the anoxic treatment.     

Fatty acid composition of lipids in the calluses of two pine species <Emphasis Type="Italic">Pinus sibirica</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

The fatty acid (FA) composition of callus lipids in two pine species, Pinus sibirica Du Tour and P. sylvestris L. was studied. Callus lipids were characterized by a high content of unsaturated FAs: 81.7% in P. sibirica and 63.2% in P. sylvestris. Among them, oleic and linoleic acids predominated (22.9 and 34.0% of total FAs in P. sibirica and 17.6 and 27.8% in P. sylvestris, respectively). Callus lipids also contained Δ5-UPIFA (unsaturated polymethyle-interrupted FAs), where pinoleic and sciadonic acids predominated. A comparison of FAs in the lipids of P. sylvestris calluses derived from needle and needle photosynthesizing tissues of this pine species showed that callus lipids were characterized by a greater diversity of Δ5-UPIFA but a lower degree of FA unsaturation and he higher level of Δ5-UPIFA.