Ammonia emission from rice leaves in relation to photorespiration and genotypic differences in glutamine synthetase activity

Background and Aims

Rice (Oryza sativa) plants lose significant amounts of volatile NH₃ from their leaves, but it has not been shown that this is a consequence of photorespiration. Involvement of photorespiration in NH₃ emission and the role of glutamine synthetase (GS) on NH₃ recycling were investigated using two rice cultivars with different GS activities.

Methods

NH₃ emission (AER), and gross photosynthesis (P_G), transpiration (Tr) and stomatal conductance (g_S) were measured on leaves of ‘Akenohoshi’, a cultivar with high GS activity, and ‘Kasalath’, a cultivar with low GS activity, under different light intensities (200, 500 and 1000 µmol m⁻² s⁻¹), leaf temperatures (27·5, 32·5 and 37·5 °C) and atmospheric O₂ concentrations ([O₂]: 2, 21 and 40 %, corresponding to 20, 210 and 400 mmol mol⁻¹).

Key Results

An increase in [O₂] increased AER in the two cultivars, accompanied by a decrease in P_G due to enhanced photorespiration, but did not greatly influence Tr and g_S. There were significant positive correlations between AER and photorespiration in both cultivars. Increasing light intensity increased AER, P_G, Tr and g_S in both cultivars, whereas increasing leaf temperature increased AER and Tr but slightly decreased P_G and g_S. ‘Kasalath’ (low GS activity) showed higher AER than ‘Akenohoshi’ (high GS activity) at high light intensity, leaf temperature and [O₂].

Conclusions

Our results demonstrate that photorespiration is strongly involved in NH₃ emission by rice leaves and suggest that differences in AER between cultivars result from their different GS activities, which would result in different capacities for reassimilation of photorespiratory NH₃. The results also suggest that NH₃ emission in rice leaves is not directly controlled by transpiration and stomatal conductance.     

Ammonia/potassium exchange in methanogenic bacteria

Methanospirillum hungatei exposed to ammonia in a K+-free buffer lost up to 98% of the cytoplasmic K+ through an ammonia/K+ exchange reaction. The exchange was immediate, and occurred in cells poisoned by air or by other metabolic inhibitors. Additions of NH4OH or various NH+4 salts (or methylamine) were most effective in causing K+ depletion in media of alkaline pH, suggesting that NH3 was the chemical species crossing the membrane. In alkaline media, the exchange reaction resulted in a dissipation of the transmembrane pH gradient (inside acidic), but had only small effects on the membrane potential until concentrations of ammonia were used above those required to abolish the K+ gradient. Through the use of NH4Cl to vary the cytoplasmic pH at a constant acidic external pH, and NH4OH to abolish the transmembrane pH gradient at various alkaline external pH values, we conclude that methanogenesis is sensitive to both the pH of the cytoplasm and the medium. Methanogenesis in Msp. hungatei and Methanosarcina barkeri was inhibited dramatically at external pH values more acidic than 6.5 or more alkaline than 7.5. Dramatic K+ depletion in response to ammonia additions at pH 8.0 occurred with Ms. barkeri, another strain of Msp. hungatei, Escherichia coli, and Bacillus polymyxa. In several other methanogens, ammonia/potassium exchange was hardly detected.     

Calculator-assisted measurements of photosynthetic,respiration and photorespiration rates in a closed gas exchange system

A closed gas exchange system has been designed for connection to the Hewlett-Packard programmable calculator controlled data acquisition system to provide a complete process of measuring and control. The system enables routine measurements of photosynthetic and dark respiration rates at different irradiances and different carbon dioxide and oxygen concentrations and leaf temperatures, and also a simple and rapid automatic control of irradiance according to the actual photosynthetic rate.     

Photosynthesis and photorespiration in algae

The CO₂ exchange of several species of fresh water and marine algae was measured in the laboratory to determine whether photorespiration occurs in these organisms. The algae were positioned as thin layers on filter paper and the CO₂ exchange determined in an open gas exchange system. In either 21 or 1% O₂ there was little difference between ¹⁴CO₂ and ¹²CO₂ uptake. Apparent photosynthesis was the same in 2, 21, or 50% O₂. The compensation points of all algae were less than 10 μl 1⁻¹. CO₂ or ¹⁴CO₂ evolution into CO₂-free air in the light was always less than the corresponding evolution in darkness. These observations are inconsistent with the proposal that photorespiration exists in these algae.     

Lead toxicity and phosphate deficiency in chlamydomonas

The addition of lead salts to phosphate-containing Chlamydomonas reinhardtii media caused precipitation of Pb₃(PO₄)₂, effectively removing phosphate from solution. The effect of Pb²⁺ on growth of Chlamydomonas in liquid cultures depended strictly on the ratio of the equivalents of Pb²⁺ to phosphate present. When the amount of Pb²⁺ approached equivalency with phosphate, cell growth was initially slow as cells adhered to the surface of the precipitated Pb₃(PO₄)₂. Later, cells grew at a normal rate, spread throughout the solution, and reached the same densities obtained in the absence of Pb²⁺. Cells did not survive when the amount of Pb²⁺ in the culture exceeded the equivalents of phosphate.

Elemental analysis showed that in the presence of equivalent Pb²⁺ and phosphate, considerable Pb²⁺ remained in solution. The concentration of dissolved Pb²⁺ did not vary significantly when the amount of Pb²⁺ added to the culture was increased slightly, from an amount which permitted growth to an amount which completely prevented growth. The concentration of phosphate was decreased to an undetectable level when the amount of Pb²⁺ approached equivalency with phosphate.

In the presence of the chelating agent nitrilotriacetic acid, higher concentrations of Pb²⁺ remained in phosphate-containing media. The chelated Pb²⁺ did not retard the growth of Chlamydomonas.

It appears that Pb²⁺ is not toxic to Chlamydomonas, but kills cells by depriving them of phosphate.

     

植物冠层与大气氨交换的研究进展

对植物与大气间NH3交换的研究进展进行了综述。大量研究表明,植物冠层能够与大气进行NH3交换．交换的大小和方向取决于细胞间隙与大气NH3分压的差异,即NH3补偿点。大部分农田植物的NH3补偿点大致在1～6nmol NH3／mol air范围内;在自然条件下,NH3补偿点可能接近气孔下腔中的NH3分压,也可能接近大气中的NH3浓度;生长在低氮输入生态系统(如沼泽地和森林)中植物的NH3补偿点几乎为零。当外界大气NH3浓度低于补偿点时,生长的植被会释放NH3：否则,植被就会吸收NH3。影响NH3补偿点的因素主要包括叶温、光照强度和空气湿度等环境因素以及质外体pH、光呼吸速率及谷氨酰胺合成酶活性等内部因素。在植物生长前期,与大气间的NH3交换以吸收为主。后期则以释放为主。植物吸收或释放NH3的大小．因研究者、试验材料和试验条件等不同而有很大差异。植物地上部分对NH3的吸收途径主要有2条,一是通过表皮层吸收;一是通过气孔吸收．通过表皮层吸收的NH3占气态NH3吸收的3％,不是主要吸收途径;NH3吸收丰要通过气孔进行,其吸收量和吸收速率取决于气孔导度、温度及光照等。目前对植物NH3释放机理的解释主要是光呼吸氮循环途径和植物衰老时的蛋白质降解途径．但有关光呼吸氮循环途径在植物体NH3挥发中的作用远末搞清。当前国内、外研究重点主要集中在植物与大气间NH3、交换的方向、强度、NH3补偿点及其影响因素,植物与大气间发生NH3交换的生理机制,人气NH3浓度增加对植物代谢和生理特征的影响等方面,而忽视了对植物生态学特征、源库特征、根系分泌物、养分利用效率和不同植物种群间竞争的影响。因此．进一步深入研究植物与大气间NH3交换。不仅可以丰富植物氮素营养理论,而且也具有十分重要的环境和生态学意义。     

Measurement of photorespiration in algae

The rates of true and apparent photosynthesis of two unicellular green algae, one diatom and four blue-green algae were measured in buffer at pH 8.0 at subsaturating concentrations of dissolved inorganic carbon (13-27 micromolar). Initial rates of depletion from the medium of inorganic carbon and ¹⁴C activity caused by the algae in a closed system were measured by gas chromatography and by liquid scintillation counting, respectively. The rate of photorespiration was calculated as the difference between the rates of apparent and true photosynthesis. The three eucaryotic algae and two blue-green algae had photorespiratory rates of 10 to 28% that of true photosynthesis at air levels of O₂. Reduction of the O₂ level to 2% caused a 52 to 91% reduction in photorespiratory rate. Two other blue-green algae displayed low photorespiratory rates, 2.4 to 6.2% that of true photosynthesis at air levels of O₂, and reduction of the O₂ concentration had no effect on these rates.     

Mutagenic effects of streptomycin in chlamydomonas

Summary Growth of a green streptomycin-resistant strain of Chlamydomonas reinhardi on a sub-lethal concentration of streptomycin on agar led to the appearance of yellow mutant cells in almost every colony. The time of appearance of the mutants varied greatly among the 9 isolates studied, each of which was selected as a single colony after repeated cloning of the parental strain. 2 isolates gave rise to colonies which responded rapidly to streptomycin (class I), 2 isolates produced yellow sub-clones as papillae only after formation of normal green colonies (class II), and 2 isolates produced stable yellow sub-clones only after a second subculture on streptomycin-agar (class III). 3 isolates were mixtures of classes II and III.The evidence that these yellow mutants arose under the mutagenic action of streptomycin is discussed in relation to the alternative possibility of their selection by the drug from a pool of pre-existing mutants. The physiological and genetic effects of streptomycin upon chlorophyll formation in Chlamydomonas are compared with reported effects of the drug upon the green flagellate, Euglena gracilis.Dedicated with appreciation and affection to Professor Dr. E. G. Pringsheim on the occasion of his 80th birthday.     

Activation and de novo synthesis of hydrogenase in chlamydomonas

Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.

Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg²⁺, Ca²⁺, and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.

The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.

     

Intraspecific measurements of photorespiration

The relative magnitudes of (a) CO₂ compensation concentration, (b) zero CO₂ intercept of the CO₂ response curve, (c) O₂ suppression of net photosynthesis, (d) differential ¹²CO₂ and ¹⁴CO₂ uptake, and (e) ¹⁴CO₂ efflux into CO₂-free air were determined in the dry bean (Phaseolus vulgaris L.) varieties Michelite-62 (M-62) and Red Kidney (RK). In comparing the two varieties for each of the above processes, there were three categories of response, M-62 > RK, M-62 = RK, and M-62 < RK. Since these processes did not give the same relative difference for the two varieties being studied, it was concluded that these phenomena cannot validly be used to estimate the magnitude of photorespiration, although they do identify its presence. The results suggest that photorespiration is but one component of O₂ inhibition of net photosynthesis and that photorespiration itself has two or more component metabolic pathways.     

Variation in photorespiration. The effect of genetic differences in photorespiration on net photosynthesis in tobacco

The hypothesis that net photosynthesis is diminished in many plant species because of a high rate of CO₂ evolution in the light has been tested further. High rates of CO₂ output in CO₂-free air in comparison with dark respiration were found in Chlamydomonas reinhardi, wheat leaves, tomato leaves, and to a lesser extent in Chlorella pyrenoidosa by means of the ¹⁴C-photorespiration assay. In tobacco leaves high photorespiration was characteristic of a standard variety, Havana Seed, and a possibly still higher rate was found in a yellow heterozygous mutant, JWB Mutant. However, the dark homozygous sibling of the latter, JWB Wild, had a low photorespiration for the tobacco species. The relative rates of photorespiration were in the same sequence when measured by the ¹⁴CO₂ released in normal air from leaf disks supplied with glycolate-1-¹⁴C in the light.

As would be predicted by the hypothesis, the maximal net rate of photosynthesis at 300 ppm of CO₂ in the air in JWB Wild leaves was greater (24%) than in Havana Seed, while JWB Mutant had less CO₂ uptake than the standard variety (21%). At 550 ppm of CO₂ the differences in net photosynthesis were not as great between the 2 siblings as at 200 ppm. The relative leaf expansion rates of seedlings of the 3 tobacco varieties in a greenhouse had the same relationship as their rates of CO₂ assimilation.

Thus within the tobacco species, as in a comparison between tobacco and maize, low photorespiratory CO₂ evolution was correlated with higher photosynthetic efficiency. Therefore it seems that increased CO₂ uptake should be achieved by genetic interference with the process of photorespiration.

     

Photosynthesis and photorespiration in whole plants of wheat

Wheat was cultivated in a small phytotronic chamber. ¹⁸O₂ was used to measure the O₂ uptake by the plant, which was recorded simultaneously with the O₂ evolution, net CO₂ uptake, and transpiration. At normal atmospheric CO₂ concentration, photorespiration, measured as O₂ uptake, was as important as the net photosynthesis. The level of true O₂ evolution was independent of CO₂ concentration and stayed nearly equal to the sum of net CO₂ photosynthesis and O₂ uptake. We conclude that at a given light intensity, O₂ and CO₂ compete for the reducing power produced at constant rate by the light reactions of photosynthesis.     

Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.     

Methyl methanesulfonate mutagenesis of synchronized chlamydomonas

Methyl methanesulfonate (MMS) mutagenesis of Chlamydomonas reinhardtii at different stages of the synchronous cell-cycle revealed the following results. (1) Induction of phenotypically distinct Mendelian (nuclear), str-50 and non-Mendelian (chloroplast) str-500P, streptomycin resistant mutants was relatively high during the first portion of the cell-cycle when chloroplast DNA replication is known to occur. (2) A second and more pronounced interval of enhanced Mendelian, str-50 mutant induction was observed near the middle of the cell-cycle when the initial stages of nuclear DNA replication occur. Induction of non-Mendelian, str-500P mutants was inconsistent during this period. (3) The incidence of mutants from a second phenotypically distinct class of non-Mendelian streptomycin-resistant mutants (str-500D) was not increased over control levels at any stage of the cell-cycle examined. It is concluded that MMS, like N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), may not be the most suitable general mutagen for this alga because its enhanced mutagenesis of cells in the nuclear S phase could result in multiple closely linked mutations.     

Arabinosyl: Hydroxyproline transferase activity in chlamydomonas reinhardtii

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardtii was found to catalyse the transfer of[³H]arabinose from UDP-arabinose to protein. Highest incorporation rates were found at 25° and pH 6.5. Hydrolytic studies on the labelled product revealed the presence of arabinose and an arabinose disaccharide in the acid hydrolysate. The transfer of arabinose to lipid was also monitored. The addition of dolichol-phosphate as an intermediate had no effect on the label incorporation into lipid. However, it had a marked inhibitory effect on the label incorporated into protein. This inhibitory effect was examined kinetically and indicated mixed-type inhibition.     

Photosynthesis and photorespiration in the genus Oryza

Photosynthetic gas exchange has been surveyed in 22 of the 23species currently placed in the genus Oryza and constitutingthe wild relatives of cultivated rice. Unimproved, wild germplasmof a number of species showed light-saturated assimilation ratesin atmospheric air at least as great as cultivars and elitebreeding material of the cultigen O. satlva. One of these specieswas O. australiensis, different accessions of which were significantly(P=0.001) superior in assimilation rate to the 0. satlva genotypestested, including representatives of the Indica, Japonica andJavanica subgroups. Amongst species, assimilation rate was correlatedpositively with light saturation and with carboxylation efficiency.The wild species fell into two distinct groupings accordingto whether they originated from sun or shade habitats, withthe higher assimilation rates being associated with the sunspecies. Assimilation rates were also higher in diploids thanin tetraploids and this was associated with the fact that allsun species are diploids and all tetraploids are shade species.The carbon dioxide compensation concentrations ranged from 28to 43µmol mol^–1 with the two lowest values (28 and32µmol mol^–1) coming from accessions of O. rufipogon.The mean value for the absolute quantum yield of photosynthesismeas ured on attached leaves was 0.060. There was a large rangein the activity of phosphoenolpyruvate (PEP) carboxylase witha number of species having rates several times those of C₃ species.Some species with the highest assimilation rates were assessedfor photorespiratory losses and these were generally around30% and similar to O. satlva cultivars. However, a range ofO. rufipogon accessions had photorespiration rates significantly(P=0.01) lower than the O. sativa genotypes tested. No speciesin the genus possessed C₄ photosynthetic metabolism though somedid overlap with compensation concentrations and phosphoenolpyruvatecarboxylase activities reported for C₃–C₄ intermediatespecies. The potential value of wild relatives to the improvementof cultivated rice is discussed. Key words: Oiyza, photorespiration, photosynthesis, rice, wild rice     

Comparative photorespiration in Amaranthus,soybean and corn

Summary A tracer technique was used to measure photorespiration in Amaranthus lividus, soybean and corn. Under a light intensity of 40 Wm^-2 (400–700 nm) efflux of tracer carbon dioxide from Amaranthus into air was comparable to that from soybean over a 30-min period and 10 times that from corn. Initial rates of efflux of tracer into air from Amaranthus were higher than from soybean and 9 times that from corn. Efflux of CO₂ from Amaranthus over 30 min in 120 Wm^-2 was only 5 times that of corn and the initial rate was only one third that of soybean. Though total efflux from soybean was similar at the two light intensities, the initial rate was slightly higher under 120 Wm^-2. For Amaranthus and soybean, pure oxygen doubled total efflux of CO₂ and substantially increased the initial rate compared with CO₂-free air whereas there was no effect on corn. A comparison of the light and dark curves suggests that light and dark respiration had different substrates. The results are interpreted in terms of the recycling of photorespiratory CO₂.