Photoactivation of Chlorophyll Synthesis and Cytochrome Oxidase Activity in Anaerobically Germinated Seedlings of Echinochloa crusgalli var. Oryzicola

When seeds of Echinochloa crusgalli var. oryzicola are germinated in dark anaerobic conditions (99.995% N₂), the seedlings do not have detectable protochlorophyll(ide). Two hours after exposure to light aerobic conditions, they begin to synthesize chlorophyll. The lag in greening is shorter in seedlings exposed to light for 24 hours before exposure to air. Seedlings maintained in light anaerobic conditions exhibit no lag in greening upon transfer to an aerobic environment. Preillumination of anaerobically grown seedlings does not result in any chlorophyll accumulation. Phytochrome is probably the receptor for photoactivation of chlorophyll synthesis, since activation is achieved by red light alone, but not by far red light or red plus far red light. The cytochrome oxidase activity in anaerobically germinated seedlings is 30% of the normal level found in aerobically grown seedlings. Preillumination was also found to activate the ability of anaerobically germinated seedlings to increase their cytochrome oxidase activity upon exposure to air.     

Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O₂.

Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O₂. Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.

Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [¹⁴C]sucrose primarily to CO₂ and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [¹⁴C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O₂. Lipids were also labeled when seeds were supplied with [¹⁴C]ethanol or [¹⁴C]acetate. Labeling experiments using the above compounds plus [¹⁴C]NaHCO₃, showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.

The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O₂. In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O₂.

     

Lipid biochemistry of echinochloa crus-galli during anaerobic germination

Seven day old seedlings of Echinochloa crus-galli var. oryzicola (Vasing) had a higher total lipid content when germinated under N₂ than in air, although ungerminated seeds contained more lipid than either seedling. The triacylglycerol pool was not depleted under anaerobiosis as it was in air and only air-grown seedlings showed a net increase in free fatty acids and polar lipids. Concentrations of most of the individual acids of the total fatty acid profile declined during germination in air and in the free acid and polar lipid fractions of these seedlings the relative proportion of polyunsaturated fatty acids increased. Compared to air-grown seedlings, ungerminated seeds and N₂-grown seedlings had a similar qualitative and quantitative lipid composition. Our results show that mobilization of storage lipids was apparently severely inhibited under anoxia. The importance of lipid metabolism to the germination and growth of Echinochloa during anoxia is discussed in terms of maintaining membrane integrity and serving (indirectly) to reoxidize pyridine nucleotides.     

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.     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

Germination and seedling growth under anaerobic conditions in Echinochloa crus-galli (barnyard grass)*

Abstract Although rice has long been recognized to be uniquely adapted for growth in low oxygen environments of flooded rice fields, rice weeds of the Echinochloa crus-galli complex appear to be at least as well specialized for germination and growth under such unusual biological conditions. Seeds of two varieties of E. crus-galli germinate and grow for prolonged periods in a totally oxygen-free environment. E. crus-galli germinates as well as rice (Oryza sativa) under a total nitrogen atmosphere and produces as large a seedling in spite of its much smaller seed size. Like rice, the seedlings of E. crus-galli are unpigmented, the primary leaves do not emerge from the coleoptile and no root growth occurs without oxygen. Of particular interest is the ultrastructure of mitochondria from anaerobically-grown seedlings. Mitochondrial profiles from the primary leaf of seedlings grown continuously in nitrogen are very similar to those grown aerobically. The size and shape of the mitochondria are similar and the cristae are numerous and normal in appearance. This is in sharp contrast to previous studies of other species which have reported that mitochondria were vesiculate and tended to lose their normal fine-structure after similar periods without oxygen. Finally, based on ultrastructure and ¹⁴C labeling studies, anaerobically-grown seedlings are highly active metabolically, which may explain, at least for E. crus-galli var. oryzicola, its ability to germinate and emerge from flooded rice fields.     

Crop mimicry in weeds

The selective forces imposed by agricultural practices have resulted in the evolution of agricultural races of weeds or agroecotypes. Some agroecotypes are intimately associated with a specific crop. Such associations can involve a system of mimicry, whereby the weed resembles the crop at specific stages during its life history and, as a result of mistaken identity, evades eradication. Mimetic forms of weeds are most likely to be selected by handweeding of seedlings or by harvesting and seed cleaning procedures. A striking example of morphological and phenological resemblance is found in the cultivated rice mimic,Echinochloa crus-galli var.oryzicola, a native of Asian rice fields but now widely distributed in rice-growing areas of the world. Comparative studies of the growth, devel-opment and patterns of phenotypic variation of cultivated rice,E. crus-galli var.oryzicola andE. crus-galli var.crus-galli demonstrate that the crop mimic is more similar to rice in many attributes than it is to its close relative. It is proposed that intense handweeding practices in Asia constitute the main selective force favoring the evolution of rice mimicry inE. crus-galli var.oryzicola.     

Anaerobic Metabolism in Germinating Seeds of Echinochloa crus-galli (Barnyard Grass) : METABOLITE AND ENZYME STUDIES

Echinochloa crus-galli, a problem weed in rice fields, has the rare ability to germinate and to grow in a totally oxygen-free environment. After 7 days growth in the light or dark under N₂, E. crus-galli var. oryzicola produces a 2- to 3-centimeter nonpigmented shoot.     

Photosynthetic Development of Anaerobically Grown Rice (Oryza sativa) after Exposure to Air

During anaerobic germination, rice produces a coleoptile devoid of carotenoid and chlorophyll. Further development and greening of the shoot occur upon exposure of the seedlings to air. In this study, a comparison was made between anaerobically (N₂) germinated rice, greened upon exposure to air, and air/dark (A/D) germinated seedlings, greened upon exposure to light. After exposure to air, N₂-grown seedlings had a 76-hour lag before net oxygen evolution occurred compared to a 6-hour lag for A/D-grown seedlings. After 98 h of greening, N₂-grown seedlings reached a rate of oxygen evolution equivalent to that of A/D-grown seedlings after 24 hours. Chlorophyll and carotenoid content showed a similar lag, but did not reach the level found in A/D-grown seedlings even after 124 hours of exposure to air. RuBPcase activity also lagged in N₂-grown seedlings, ultimately reaching greater values than in the `greened' A/D-grown seedlings. Phosphoenolpyruvate carboxylase activity was constant and low in all treatments except for a transient increase after 24 hours of greening of the N₂-grown seedlings.     

ENZYME LOCALIZATION IN THE ANAEROBIC MITOCHONDRIA OF ASCARIS LUMBRICOIDES

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg⁺⁺-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.     

Cytochromes of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Rice seeds were germinated for up to 5 days under water (submerged)and some for another day in air (air-adapted). Control seedswere germinated for 6 days throughout in air. Low-temperaturedifference spectra of shoot mitochondria were compared amongthese three types of seedlings. All cytochromes found in theaerobic seedlings were present in the submerged seedlings. However,there were some differences in the cytochromes b₅₅₃ and c ofthese two types of seedlings. The cytochrome aa₃ peak heightand cytochrome oxidase activity per mitochondrial protein increased1.6- and 2.8-fold, respectively, during air adaptation. Slightlyhigher concentrations of the b-type cytochromes than found inair-adapted mitochondria were already present in submerged mitochondria.The computed difference between the dithionite-reduced differencespectra of mitochondria from submerged seedlings before andafter air adaptation, showed that cytochromes aa₃ and c hadincreased more than cytochrome b₅₅₇ during air adaptation. (Received November 16, 1987; Accepted March 16, 1988)     

Respiratory Properties of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Respiratory activities were compared among rice seedlings germinated in air for 6 days (aerobic seedlings), those germinated under water for 5 days (submerged seedlings), and those grown in air for 1 day after 5 days' submerged germination (air-adapted seedlings). The respiratory activity of the submerged seedlings increased rapidly on transfer to air and reached a plateau at 16 hours in air. Respiration of the submerged seedlings was as sensitive to cyanide as those of aerobic and air-adapted seedlings. 2,4-Dinitrophenol had no effect on the respiration of the submerged seedlings, but stimulated those of the other two types of seedlings. Mitochondria from three types of seedlings did not differ in the ADP/O ratio and the respiratory control ratio (RCR) when succinate was oxidized. However, mitochondria from submerged seedlings (submerged mitochondria) showed poor RCR of about unity when malate was oxidized. Both the rate of succinate oxidation and succinate dehydrogenase activity were low in submerged mitochondria, but increased during air adaptation. Although submerged mitochondria oxidized malate very slowly, this activity increased after exposure to air without any increase in malate dehydrogenase activity. When NAD⁺ was added to submerged mitochondria, oxidation of malate was restored to the level of the aerobic controls. Addition of NAD⁺ enhanced the state 3 rate in submerged mitochondria, and RCR recovered to nearly the same value as that of the aerobic controls. Similar effects of NAD⁺ on 2-oxoglutarate oxidation were observed. All these defects in submerged mitochondria were repaired during air adaptation. These results suggest that NAD⁺-linked substrate oxidation was low in submerged mitochondria because of NAD⁺ deficiency, and that the oxidation increased with an increasing level of NAD⁺ during air adaptation.     

Evidence for plantlet regeneration via somatic embryogenesis in the grasses Echinochloa muricata and E. crus-galli var. Oryzicola

Explants for tissue culture were derived from mesocotyl plate tissue of Echinocha crus-galli var. oryzicola and E. muricata seedlings germinated under anaerobic conditions. Callus was initiated in the dark under aerobic conditions on a modified Murashige and Skoog medium plus 10 or 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l of 6-benzyl-aminopurine (BAP). Transfer of this callus tissue into the light on MS medium containing low auxin (≤ 5 mg/l) readily resulted in the formation of green plantlets. Scanning electron microscopic observations revealed that regeneration occured through the formation of somatic embryros. Capacity for regeneration is maintained after repeated callus subculture. This regenerative capacity via somatic embyros provides a valuable research system for continuing the study of the metabolism and developmental physiology of Echinochloa.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

The influence of aging conditions on the short term growth of green pea stem segments

Green pea (Pisum sativum L. var. Alaska) stem segments that were aged in buffer responded differently after aging depending on whether they were floating or submerged, or bubbled with air or N₂. Segments aged anaerobically for only 1 to 2 hours at 23 C responded to subsequent aerobic conditions by elongating more rapidly than aerobically aged sections. Longer periods of anaerobic treatment (up to 5 hours at 23 C) caused sections to exhibit an auxin-insensitive growth lag and reversible shrinkage. The shrinkage accelerated upon return to aerobic conditions but reversed after 1 to 2 hours. Green pea stem segments therefore require vigorous aeration during aging and growth measurements.     

Germination of Echinochloa crus-galli (Barnyard Grass) Seeds under Anaerobic Conditions : Respiration and Response to Metabolic Inhibitors

Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO₂ in Echinochloa and rice is lower and the peak rate of CO₂ evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO₂] N₂/[CO₂] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.

Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N₂, but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed.

     

Biochemical and evolutionary aspects of anaerobically functioning mitochondria

Mitochondria are usually considered to be the powerhouses of the cell and to be responsible for the aerobic production of ATP. However, many eukaryotic organisms are known to possess anaerobically functioning mitochondria, which differ significantly from classical aerobically functioning mitochondria. Recently, functional and phylogenetic studies on some enzymes involved clearly indicated an unexpected evolutionary relationship between these anaerobically functioning mitochondria and the classical aerobic type. Mitochondria evolved by an endosymbiotic event between an anaerobically functioning archaebacterial host and an aerobic alpha-proteobacterium. However, true anaerobically functioning mitochondria, such as found in parasitic helminths and some lower marine organisms, most likely did not originate directly from the pluripotent ancestral mitochondrion, but arose later in evolution from the aerobic type of mitochondria after these were already adapted to an aerobic way of life by losing their anaerobic capacities. This review will focus on some biochemical and evolutionary aspects of these fermentative mitochondria, with special attention to fumarate reductase, the synthesis of the rhodoquinone involved, and the enzymes involved in acetate production (acetate : succinate CoA-transferase and succinyl CoA-synthetase).     

Mitochondrial enzymes in aerobically and anaerobically germinated seedlings of Echinochloa and rice

Activities of tricarboxylic acid (TCA) cycle enzymes in seedlings of barnyard grass (Echinochloa phyllopogon (Stapf.) Koss) and rice (Oryza sativa L.) germinated under aerobic and anaerobic conditions were investigated. In E. phyllopogon, development of TCA-cycle enzyme activities during 10 d of anoxia generally paralleled those in air, although at lower rates. After 5 d, E. phyllopogon seedlings germinating under N₂ exhibited 50–80% of the activity of seedlings grown in air, except for 2-oxoglutarate dehydrogenase (EC 1.2.4.2) and fumarate reductase (EC 1.3.1.6) which exhibited only 25–35% of aerobic activity. In anaerobically germinated rice, development of TCA-cycle enzyme activities also paralleled those in air except for aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.41), and 2-oxoglutarate dehydrogenase. Those enzymes did not increase in activity under anoxia. Development of maximum enzyme activities generally occurred more rapidly and persisted longer in E. phyllopogon compared to rice. The data indicate that mitochondria of E. phyllopogon function better during anaerobiosis than those of rice and this factor may contribute to the successful biochemical strategy of this weed in rice paddies throughout the world.Abbreviation TCA tricarboxylic acid This work was supported by U.S. Department of Agriculture Competitive Research grant No. 87-CRCR1-2595 and a Herman Frasch Foundation grant in Agricultural Chemistry to R.A.K.     

Development of Respiration and Mitochondria in Mucor genevensis After Anaerobic Growth: Absence of Glucose Repression

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO(4) fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.     

Metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobacter sphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2–3 times lower in the cells of the M variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa ₃, acting as cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient at the cytochrome aa ₃ level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.