Distribution of radioactivity in carbon atoms of malic acid formed during light-enhanced dark 14CO2-fixation in maize leaves

Most of the radioactivity incorporated into malic acid duringlight-enhanced dark ¹⁴CO₂-fixation was found in C-4, supportingour conclusion that phosphoenolpyruvic acid serves as a primaryacceptor of ¹⁴CO₂ to form the C₄ acid. ¹This work was reported at the 13th Annual Meeting of JapaneseSociety of Plant Physiologists, April, 1972. (Received January 6, 1973; )     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

High CO2 partial pressure effects on dark and light CO2 fixation and metabolism in Vicia faba leaves

Stomatal opening on Vicia faba can be induced by high CO₂ partial pressures (10.2%) in dark as well as in light. Stomatal aperture was measured in both cases with a hydrogen porometer. The distribution of ¹⁴C among early products of photosynthesis was studied. Comparisons are made with carboxylations occurring when stomata were open in the dark with CO₂-free air and in light with 0.034% CO₂. Results showed that in high CO₂ partial pressure in light, less radioactivity was incorporated in Calvin cycle intermediates and more in sucrose. carboxylations and photorespiration seemed to be inhibited. In the dark in both CO₂ conditions, ¹⁴C incorporation was found in malate and aspartate but also in serine and glycerate in high CO₂ conditions. In light these changes in metabolic pathways may be related with the deleterious effects recorded on leaves after long-term expositions to high partial pressure of CO₂.Abbreviations DHAP dihydroxyacetone phosphate - PEP phosphonenolpyruvate - PEPCK phosphonenolpyruvatecarboxykinase - PGA 3-phosphoglyceric acid - RUBPc ribulose 1,5-bisphosphate carboxylase     

Diurnal fluctuation of light and dark CO2 fixation in peeled and unpeeled leaves of Bryophyllum daigremontiana

Diurnal fluctuation of light and dark CO₂ fixation in peeledand unpeeled leaves of Bryophyllum daigremontiana was examined.A distinct difference in light CO₂ fixation was observed inunpeeled leaves but not in peeled ones. No measurable differencein dark CO₂ fixation was observed in either type. These resultsindicate that the leaves of CAM plants have a high capacityfor CO₂ fixation in the daytime, but it is suppressed by theclosing of the stomata. Also, the rapid depression of CO₂ uptakewhen the illumination was directed at on dark acidified leavescould be prevented by peeling off the epidermis. The net photosyntheticCO₂ uptake in peeled leaves was 77 µmoles/mg chllrophyll/hrin the 3rd leaf and 62 in the 4th leaf. (Received August 7, 1978; )     

Prior illumination and the respiration of maize leaves in the dark

The course of respiration of attached maize (Zea mays L.) leaves was measured by infrared gas analysis of CO₂ efflux in the dark following illumination in atmospheres of 300 microliters of CO₂ per liter of air, CO₂-free air, and CO₂-free N₂ containing 400 microliters of O₂ per liter. CO₂ efflux from control leaves started 3 to 4 minutes after darkening, increased to a maximum after about 20 minutes, and returned to a steady minimum after 2 to 3 hours. Respiration was quantitatively related to prior illumination, independent of net CO₂ fixation in the light, and depressed by N₂. Light, but not air, was required to produce a substrate for respiration in the subsequent dark period; air was required for oxidation of the substrate to CO₂. The stimulation of respiration by prior illumination in maize leaves differs in its slower onset and greater duration from the postillumination burst of photorespiration.     

Effect of leaf age on light and dark 14CO2 fixation in a CAM plant, Bryophyllum calycinum

Leaves of different ages from B. calycinum were exposed to ¹⁴CO₂in light during day and night. The labelling pattern on thechromatogram differed with leaf age. Young leaves had similarpatterns to those of C3 plants during both day and night. Matureleaves showed high incorporation of ¹⁴C into C4 acids, especiallyat night. In contrast, no significant difference with leaf agewas observed in the pattern of dark ¹⁴CO₂ fixation products.Study of the enzyme activity and the content of titratable acidat each leaf age suggested that high incorporation of ¹⁴C inC4 acids during the night was due to the simultaneous absorptionof CO₂ by both enzymes RuDPcarboxylase and PEPcarboxylase. (Received November 24, 1977; )     

Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd

The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO₂; in the absence of CO₂ dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO₂. Dark CO₂ fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO₂ in the dark; in the lichen most dark CO₂ fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of ¹⁴CO₂ in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO₂ in the gas phase. Exogenous NH ₄ ⁺ inhibited nitrogenase activity by cephalodia in the dark especially in the absence of CO₂ but had no effect in the light. The overall data suggest that in the lichen dark CO₂ fixation by the fungus may provide carbon skeletons which accept NH ₄ ⁺ released by the cyanobacterium and that in the absence of CO₂, NH ₄ ⁺ directly, or indirectly via a mechanism which involves glutamine synthetase, inhibits nitrogenase activity.Abbreviations CP carbamoyl phosphate - EDTA ethylenedi-amine tetraacetic acid - PEP phosphoenolpyruvate - RuBP ribulose 1,5 bisphosphate     

Photosynthetic 14CO2 fixation products in altitudinal ecotypes of Trifolium repens L. with different temperature requirements

Summary Two Trifolium repens clones from natural meadows at 600 m and 2030 m above sea level, and with differing dependence on temperature of their rate of apparent photosynthesis, were grown under controlled environments. Radioactive products in detached leaves were examined after 20 and 40 s periods of steady state photosynthesis in ¹⁴CO₂ at 3° C and 24° C. Glycine and serine were hardly labeled at 3° C. At 24° C, the leaves of the alpine clone showed significantly, (P<0.025) more activity in these amino acids than those from the low altitude clone. It is suggested that the alpine clone has a higher photorespiration. This is supported by the labeling pattern of glucose, fructose, sucrose, and glucose-6-phosphate.The high altitude clone requires lower temperatures for photosynthesis than the low altitude clone. It is suggested, that this is caused by its higher photorespiration, which reduces net photosynthesis at high temperatures. The lower photorespiration activity of the low altitude clone can be interpreted as an adaptation to its warmer habitat.     

Light and dark CO2 fixation in Clusia uvitana and the effects of plant water status and CO2 availability

Summary In well-watered plants of Clusia uvitana, a species capable of carbon fixation by crassulacean acid metabolism (CAM), recently expanded leaves gained 5 to 13-fold more carbon during 12 h light than during 12 h dark periods. When water was withheld from the plants, daytime net CO₂ uptake strongly decreased over a period of several days, whereas there was a marked increase in nocturnal carbon gain. Photosynthetic rates in the chloroplasts were hardly affected by the water stress treatment, as demonstrated by measurements of chlorophyll a fluorescence of intact leaves, indicating efficient decarboxylation of organic acids and refixation of carbon in the light. Within a few days after rewatering, plants reverted to the original gas exchange pattern with net CO₂ uptake predominantly occurring during daytime. The reversible increase in dark CO₂ fixation was paralleled by a reversible increase in the content of phosphoenolpyruvate (PEP) carboxylase protein. In wellwatered plants, short-term changes in the degree of dark CO₂ fixation were induced by alterations in CO₂ partial pressure during light periods: a decrease from 350 to 170 bar CO₂ caused nocturnal carbon gain, measured in normal air (350 bar), to increase, whereas an increase to 700 bar CO₂, during the day, caused net dark CO₂ fixation to cease. The increased CAM activity in response to water shortage may, at least to some extent, be directly related to the reduced carbon gain during daytime.     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Products of CO2 fixation and 14C labelling pattern of alanine in Methanobacterium thermoautotrophicum pulse-labelled with 14CO2

The incorporation of ¹⁴CO₂ by an exponentially growing culture of the autotrophic bacterium Methanobacterium thermoautotrophicum has been studied. The distribution of radioactivity during 2s–120s incubation periods has been analyzed by chromatography and radioautography. After a 2 s incubation most of the radioactivity of the ethanolsoluble fraction was present in the amino acids alanine, glutamate, glutamine and aspartate, whereas phosphorylated compounds were only weakly labelled. The percentage of the total radioactivity fixed, which was contained in the principal early labelled amino acid alanine, increased in the first 20 s and only then decreased, indicating that alanine is derived from primary products of CO₂ fixation.The labelling patterns of alanine produced during various incubation times have been determined by degradation. After a 2 s ¹⁴CO₂ pulse, 61% of the radioactivity was located in C-1, 23% in C-2, and 16% in C-3. The results are consistent with the operation of a previously proposed autotrophic CO₂ assimilation pathway which involves the formation of acetyl CoA from 2 CO₂ via one-carbon unit intermediates, followed by the reductive carboxylation of acetyl CoA to pyruvate.     

The effect of oxygen on 14CO2 fixation in mesophyll cells isolated from Digitaria sanguinalis (L.) Scop. leaves

Mesophyll cells were isolated from fully-expanded leaves of $\text{Digitaria sanguinalis}$ (L.) Scop. by a combined maceration-filtration technique. In the presence of pyruvate, photosynthetic ¹⁴CO₂ uptake in the isolated cells was not inhibited by atomospheric levels of oxygen. In contrast, superatmospheric levels of oxygen substantially inhibited the light-dependent fixation of ¹⁴CO₂. These oxygen effects are similar to those observed with intact C4 leaves and suggest that the lack of inhibition of C4 photosynthesis by atmospheric levels of oxygen results from the relative oxygen-insensitivity of the phosphopyruvate carboxylase-CO₂ pump in the mesophyll.     

Chlorophyll a and b biosynthesis in the dark in etiolated leaves infiltrated by exogenous chlorophyllide a]

Exogenous chlorophyllide a was introduced into etiolated rye leaves by the vacuum-infiltration technique. Appearance and accumulation of chlorophylls a and b within the leaves are observed during continued darkening, protochlorophyllide photoreduction being avoided. The pigments are identified by the solubility in petroleum ether, paper chromatograms, the fluorescence maxima, the peculiarities of exciting light 430 and 460 nm effects on fluorescence intensity, the specific interaction with hydrochloric hydroxylamine. The conclusion is made that before illumination etioplasts already contain enzyme systems and substrates which provide esterification of chlorophyllide a to chlorophyll a and conversion of chlorophyll a into chlorophyll b.     

Ontogeny of the vascular bundles and contiguous tissues in the maize leaf blade

The patterns of initiation and early development of the minor and major veins in the flat portion of the leaf blade of maize (Zea mays L.) follow similar patterns. The veins and their associated bundle sheath cells commonly arise from cell assemblages derived from a single cell lineage, or longitudinal file of cells, rather than from two “half vein units” derived from different cell lineages. In addition, apparently, none of the vascular cells derived from the procambium is directly related ontogenetically to a bundle sheath cell. In veins derived from larger cell assemblages, the lateral bundle sheath cells are more closely related ontogenetically to the mesophyll cells, which are derived from the ground meristem, than to the vascular cells, which are derived from procambium. The bundle sheath cells, accordingly, are interpreted as being ground meristem in origin.     

胡杨异形叶光合作用对光强与CO2浓度的响应

胡杨(Populus euphratica)叶形多变, 随个体生长发育, 植株出现条形、卵形和锯齿阔卵形叶。在新疆塔里木河上游人工胡杨林内选择具有此3种叶形的成年标准株, 将枝条拉至同一高度, 通过活体测定, 比较其光合作用-光与CO₂响应及叶绿素荧光响应特征。结果表明: 胡杨异形叶光合速率对光强/CO₂浓度与电子传递速率对光强的响应曲线均可用直角双曲线修正模型来拟合, 得出的主要光合参数与实测值较吻合。胡杨卵形叶、锯齿阔卵形叶光合速率-光响应参数与生化参数及快速光响应参数与条形叶差异显著, 而光合速率-CO₂响应参数则无显著差异。胡杨异形叶CO₂饱和浓度下的最大净光合速率(P_nmax)较饱和光强下的P_nmax高, 表明胡杨强光下光合速率在很大程度上受CO₂供应和1,5-二磷酸核酮糖(RuBP)再生能力的限制。卵形叶、锯齿阔卵形叶的初始量子效率(α)、初始羧化效率(CE)、P_nmax、光合能力(A_max)与最大羧化速率(V_cmax)均显著高于条形叶; 锯齿叶光饱和点(LSP)、最大电子传递速率(ETR_max)与光呼吸速率(R_p)高于卵形叶, 条形叶光补偿点(LCP)与LSP、α、CE最低。表明荒漠干旱环境下胡杨锯齿叶最耐强光, 高R_p可能是其耗散过剩光能、保护光合机构免于强光破坏的重要途径; 卵形叶高的α、CE、磷酸丙糖利用效率(TPU)、PSII实际光化学效率(Φ_PSII)与低LCP及叶氮分配策略是其保持高光合速率的原因; 条形叶Φ_PSII、ETR、P_n低, 因其制造光合产物不足而难以满足树体生长逐渐减少并处于树冠下部。可见, 胡杨条形叶光合效率低、抗逆性差, 主要以维持生长为主; 随着树体长大, 条形叶难以适应荒漠环境来维系其生长, 出现了卵形叶; 卵形叶光合效率高, 易于快速积累光合产物而加快树体生长, 但其LSP低和耐光抑制能力弱, 逐渐被更耐强光、高温与大气干旱的锯齿叶所取代, 从而使胡杨在极端逆境下得以生存, 这是胡杨从幼苗到成年叶形变化及异形叶着生在树冠不同高度的原因。     

Pod photosynthesis and seed dark CO2 fixation support oil synthesis in developingBrassica seeds

Rate of photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were determined in pods (siliqua), whereas rate of dark CO₂ fixation, oil content and activities of enzymes involved in dark CO₂ metabolism were measured in seeds ofBrassica campestris L. cv. Toria at different stages of pod/seed development. The period between 14 and 35 days after anthesis corresponded to active phase of seed development during which period, seed dry weight and oil content increased sharply. Rate of pod photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were maximum in younger pods but sufficiently high levels were retained up to 40 days after anthesis. The rate of dark¹⁴CO₂ fixation in seeds increased up to 21 days after anthesis and declined thereafter but maintaining sufficiently high rates till 35 days after anthesis. Similarly various enzymes viz., phosphoenolpyruvate carboxylase, NAD⁺-malate dehydrogenase and NADP⁺-malic enzyme, involved in dark CO₂ metabolism retained sufficient activities during the above period. These enzyme activities were more than adequate to maintain the desired supply of malate which mainly arises from dark CO₂ fixation in seeds and further translocated to leucoplasts for onward synthesis of fatty acids. Enzyme localization experiments revealed phosphoenolpyruvate carboxylase and enzymes of sucrose metabolism to be present only in cytosol, whereas enzymes of glycolysis were present both in cytosolic and leucoplastic fractions. These results indicated that oil synthesis in developingBrassica seeds is supported by pod photosynthesis and dark CO₂ fixation in seeds as the former serves as the source of sucrose and the latter as a source of malate     

Size-fractionated biomass, photosynthesis and dark CO2 fixation in a tropical oceanic environment

This study examines the spatial distribution and size structureof phytoplankton biomass and productivity in relation to thevertical structrure of the Andaman Sea (northeastern IndianOcean). This region was characterized by low concentrationsof nutrients and high levels of insolation. Nitrogen availabilityappeared to control overall productivity with nitrate-based‘new’ production accounting for 8–24% of thetotal primary production. Euphotic column chlorophyll (chl a)averaged 52.5 mg m^–2 of which a major portion was locatedas a subsurface chl a maximum (SCM) at