Metabolism of Oat Leaves during Senescence : VII. The Interaction of Carbon Dioxide and Other Atmospheric Gases with Light in Controlling Chlorophyll Loss and Senescence

In air largely freed from CO₂, senescence of isolated oat (Avena sativa cv Victory) seedling leaves is no longer prevented by white light; instead, the leaves lose both chlorophyll and protein as rapidly as in the dark. Senescence in light is also accelerated in pure O₂, but it is greatly delayed in N₂; 100% N₂ preserves both protein and chlorophyll in light and in darkness. In light in air, most of the compounds tested that had previously been found to delay or inhibit senescence in darkness actually promote the loss of chlorophyll, but they do not promote proteolysis. Under these conditions, proteolysis can therefore be separated from chlorophyll loss. But in light minus CO₂, where chlorophyll loss is rapid in controls, two of these same reagents prevent the chlorophyll loss. Unlike the many reagents whose action in light is thus the opposite of that in darkness, abscisic acid, which promotes chlorophyll loss in the dark, also promotes it in light with or without CO₂. Kinetin, which prevents chlorophyll loss in the dark, also prevents it in light minus CO₂. In general, therefore, the responses to light minus CO₂ are similar to the responses to darkness, and (with the exception of abscisic acid and kinetin) opposite to the response to light in air.     

Trends in carbohydrate depletion, respiratory carbon loss, and assimilate export from soybean leaves at night

To evaluate assimilate export from soybean (Glycine max [L.] Merrill) leaves at night, rates of respiratory CO₂ loss, specific leaf weight loss, starch mobilization, and changes in sucrose concentration were measured during a 10-hour dark period in leaves of pod-bearing `Amsoy 71' and `Wells II' plants in a controlled environment. Lateral leaflets were removed at various times between 2200 hours (beginning dark period) and 0800 hours (ending dark period) for dry weight determination and carbohydrate analyses. Respiratory CO₂ loss was measured throughout the 10-hour dark period. Rate of export was estimated from the rate of loss in specific leaf weight and rate of CO₂ efflux. Rate of assimilate export was not constant. Rate of export was relatively low during the beginning of the dark period, peaked during the middle of the dark period, and then decreased to near zero by the end of darkness. Rate of assimilate export was associated with rate of starch mobilization and amount of starch reserves available for export. Leaves of Amsoy 71 had a higher maximum export rate in conjunction with a greater total change in starch concentration than did leaves of Wells II. Sucrose concentration rapidly declined during the first hour of darkness and then remained constant throughout the rest of the night in leaves of both cultivars. Rate of assimilate export was not associated with leaf sucrose concentration.     

Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves

Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO₂ uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de-epoxidation), as well as nonphotochemical quenching (q_NP), and the rate constant of radiationless energy dissipation (k_D). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO₂ uptake differed widely and light saturation of CO₂ uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO₂ uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q_NP, increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO₂ uptake was approached, q_NP showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k_D, which opens the possibility that zeaxanthin is related to the rapidly relaxing “high-energy-state quenching” in leaves.     

The H+-Sucrose Cotransporter NtSUT1 Is Essential for Sugar Export from Tobacco Leaves

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H⁺-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed ¹⁴CO₂ and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO₂ fixation even after a 24-h chase. The ¹⁴C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.     

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.

     

Effect of carbon dioxide and light on ethylene production in intact sunflower plants

High CO₂ concentration (0.5%) increased the rate of ethylene production, measured in a continuous flow system, in intact sunflower (Helianthus annuus L.) plants. However, the rate of ethylene production subsided to near control levels after approximately 24 hours. The effect of high CO₂ could only be observed in light. Although high CO₂ concentration had no effect on the rate of ethylene production in darkness, prolonged exposure (approximately 16 hours) of plants to high CO₂ in the dark prevented the increase in ethylene production when the plants were exposed to light and high CO₂.     

Inhibition of photosynthesis by carbohydrates in wheat leaves

The rate of net CO₂ assimilation of mature wheat (Triticum aestivum L.) leaves in ambient air (21% O₂, 340 microbars CO₂) declined with time of illumination at temperatures lower than 25°C, but not at higher temperatures, and the rate of decline increased when maintained in air with higher CO₂ concentration (700-825 microbars). In this latter case, the decline in the rate of net CO₂ assimilation also occurred at high temperatures. Stomatal conductance also declined with time in some cases and stomata became more sensitive to CO₂, but this was not the primary cause of the decrease in CO₂ assimilation because internal partial pressure of CO₂ remained constant. Treatments which reduced the rate of translocation (e.g. lower temperatures, chilling the base of the leaf) produced a marked decline in CO₂ assimilation of leaves in atmospheric and high CO₂ concentrations. The decreased net CO₂ assimilation was correlated with carbohydrate accumulation in each case, suggesting end product inhibition of photosynthesis. Analysis of CO₂ assimilation in high carbohydrate leaves as a function of intercellular CO₂ partial pressure showed reduction in the upper part of the curve. The initial slope of this curve, however, was not affected. Photosynthetic rates in the upper part of this curve generally recovered after a short period in darkness in which carbohydrates were removed from the leaf. The stimulation of net CO₂ assimilation by 2% O₂ (Warburg effect), and the apparent quantum yield, decreased after several hours of light.     

Diurnal and seasonal changes in the intensity of photosynthesis in stems of lilac (Syringa vulgaris L.)

It has been demonstrated that during the whole year the stems are photosyntheticaly active and capable of assimilating atmospheric CO₂. The intensity of photosynthesis varies. During the vegetation period the registered net photosynthesis lasted up to 13 hours per day, and in the leafless period for 2–3 hours a day. Photosynthesis was registered also at temperatures below zero (−3 °C) as a reduced CO₂ evolution in light in comparison with darkness. The maximal net photosynthesis values during the vegetation period amounted to 6 up 8 μmol (CO₂)·m⁻²·s⁻¹, and in the leafless period 0.5 – 1 μmol (CO₂)·m⁻²·s⁻¹, and they were close to being up to twice as big as the values obtained of darkness respiration. An increase of the photosynthetic activity of stems preceded the spring development of the leaves.     

The state of activation of ribulose-1,5-bisphosphate carboxylase in wheat leaves

In light and in darkness, exposure of leaf segments to CO₂-free atmospheres caused a marked reduction in extractable RuBP carboxylase activity. By contrast, darkness caused a relatively small decrease in carboxylase activity in extracts from leaf segments kept in air containing CO₂. Recovery of carboxylase activity in leaves during illumination in air after exposure to CO₂-free conditions paralleled recovery of capacity for photosynthesis; in darkness recovery of carboxylase activity in leaves was slower than in the light. Extracts from leaves exposed to CO₂-free conditions recovered activity when provided with CO₂ and Mg²⁺; there were clearly, however, substances in the extracts that modified the activity achieved and caused anomalous decreases and increases with time after extraction. Studies of the effect of orthophosphate on the activity of purified wheat carboxylase in vitro were consistent with the view that many of the effects observed on the activity of crude leaf extracts were due to orthophosphate content.     

Evaluation of the light/dark C assay of photorespiration: tobacco leaf disk studies with glycidate and glyoxylate

Preincubation of illuminated tobacco (Nicotiana tabacum L.) leaf disks in glycidate (2,3-epoxypropionate) or glyoxylate inhibited photorespiration by about 40% as determined by the ratio of ¹⁴CO₂ evolved into CO₂-free air in light and in darkness. However, under identical preincubation conditions used for the light/dark ¹⁴C assays, the compounds failed to reduce photorespiration or stimulate net photosynthesis in tobacco leaf disks based on other CO₂ exchange parameters, including the CO₂ compensation concentration in 21% O₂, the inhibitory effect of 21% O₂ on net photosynthesis in 360 microliters per liter of CO₂ and the rate of net photosynthetic ¹⁴CO₂ uptake in air.

The effects of both glycidate and glyoxylate on the ¹⁴C assay are inconsistent with other measures of photorespiratory CO₂ exchange in tobacco leaf disks, and thus these data question the validity of the light to dark ratio of ¹⁴CO₂ efflux as an assay for relative rates of photorespiration (Zelitch 1968, Plant Physiol 43: 1829-1837). The results of this study specifically indicate that neither glycidate nor glyoxylate reduces photorespiration or stimulates net photosynthesis by tobacco leaf disks under physiological conditions of pO₂ and pCO₂, contrary to previous reports.

     

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m⁻² s⁻¹ was followed, the slow increase in CO₂ assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO₂ assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO₂ assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.     

Effects of Temperature on Photosynthesis and CO(2) Evolution in Light and Darkness by Green Leaves

Using an open and a closed system of gas analysis, it was found that CO₂ evolution in light and in darkness from plant leaves (sunflower, soybean, watermelon, eggplant, and jackbean) have a different response to temperature. While the rate of CO₂ evolution in light increased with increasing temperature from 17 to 35° and then declined, the rate of CO₂ evolution in darkness increased continuously up to 40°. The rate of CO₂ evolution in light was affected by light intensity. At 1800 ft-c and below 35° the rate of CO₂ evolution in light was greater than in darkness, but above 35° it became lower than in darkness. The Q₁₀ for CO₂ evolution in light was consistently lower than that in darkness.     

Effect of toxaphene on carbon dioxide assimilation and translocation in avena secale

In susceptible oat, toxaphene inhibits photosynthetic electron flow and concomitant ATP synthesis. Although the rate of ¹⁴CO₂ assimilation is apparently not affected markedly there is an increase in dry weight of leaves contacting the pesticide. The labelling patterns in leaf sections exposed to ¹⁴CO₂ are similar for both toxaphene-treated and untreated seedlings. However, if given a period in darkness before extraction it is evident that assimilation products in leaf sections from toxaphene-treated leaves remain as small M, materials, including substantial amounts of sugars, whereas in untreated controls these were converted to polymeric materials. In toxaphene-treated seedlings the translocation of assimilation products to the roots is decreased and sucrose accumulates in the leaves.     

Effects of Metabolic Inhibitors on the Rates of CO(2) Evolution in Light and in Darkness by Detached Spruce Twigs, Wheat, and Soybean Leaves

Detached spruce twigs, wheat and soybean leaves were infiltrated with various metabolic inhibitors, placed in a closed system in CO₂-free air and the amounts of CO₂ evolved in either light or darkness were determined with an infra-red CO₂ analyzer. In light, metabolic inhibitors always greatly suppressed evolution of CO₂, the magnitude of suppression varying between 50 to 80% of that without an inhibitor. This depressing effect became less pronounced with increasing oxygen. In darkness, metabolic inhibitors sometimes suppressed and sometimes stimulated CO₂ evolution. These observations have been taken as further support for a conclusion made earlier, that evolution of CO₂ in light and darkness is not the same process.     

Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves

The in vivo oxidation of the C₄ and C₅ of 5-aminolevulinic acid (ALA) to CO₂ has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of ¹⁴CO₂ evolution from leaves fed [4-¹⁴C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of ¹⁴CO₂ evolution from leaves fed [5-¹⁴C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C₄ is oxidized to CO₂ via the tricarboxylic acid cycle to a greater extent than is the C₅.     

Relationship between Photosynthesis and Respiration: The Effect of Carbohydrate Status on the Rate of CO(2) Production by Respiration in Darkened and Illuminated Wheat Leaves

The rate of dark CO₂ efflux from mature wheat (Triticum aestivum cv Gabo) leaves at the end of the night is less than that found after a period of photosynthesis. After photosynthesis, the dark CO₂ efflux shows complex dependence on time and temperature. For about 30 minutes after darkening, CO₂ efflux includes a large component which can be abolished by transferring illuminated leaves to 3% O₂ and 330 microbar CO₂ before darkening. After 30 minutes of darkness, a relatively steady rate of CO₂ efflux was obtained. The temperature dependence of steady-state dark CO₂ efflux at the end of the night differs from that after a period of photosynthesis. The higher rate of dark CO₂ efflux following photosynthesis is correlated with accumulated net CO₂ assimilation and with an increase in several carbohydrate fractions in the leaf. It is also correlated with an increase in the CO₂ compensation point in 21% O₂, and an increase in the light compensation point. The interactions between CO₂ efflux from carbohydrate oxidation and photorespiration are discussed. It is concluded that the rate of CO₂ efflux by respiration is comparable in darkened and illuminated wheat leaves.     

Postillumination respiration of maize in relation to oxygen concentration and glycolic Acid metabolism

Prior illumination in CO₂-free air enhances a respiration from maize (Zea mays L.) leaves different in onset and duration from the postillumination burst of photorespiration. The course of respiration after brief illumination of attached leaves was measured as CO₂ efflux in darkness into CO₂-free atmospheres with four O₂ concentrations. The peak of CO₂ efflux following illumination was suppressed by 2.23% O₂, was completely eliminated by 0.04% O₂, and was not stimulated by 40% O₂ compared with air. Compared with air, steady dark respiration was suppressed by 0.04% O₂ but was not affected by 2.23% nor 40% O₂. Excision and subsequent uptake of distilled water through the vascular system nearly eliminated the enhanced respiration.     

Effect of Oxygen on Photosynthesis, Photorespiration and Respiration in Detached Leaves. II. Corn and other Monocotyledons

The effect of O₂ on the CO₂ exchange of detached leaves of corn (Zea mays), wheat (Triticum vulgare), oats (Avena sativa), barley (Hordeum vulgare), timothy (Phleum pratense) and cat-tail (Typha angustifolia) was measured with a Clark oxygen electrode and infrared carbon dioxide analysers in both open and closed systems.

Corn leaves did not produce CO₂ in the light at any O₂ concentration, as was shown by the zero CO₂ compensation point and the absence of a CO₂ burst in the first minute of darkness. The rate of photosynthesis was inhibited by O₂ and the inhibition was not completely reversible. On the other hand, the steady rate of respiration after a few minutes in the dark was not affected by O₂.

These results were interpreted as indicating the absence of any measurable respiration during photosynthesis. Twelve different varieties of corn studied all responded to O₂ in the same way.

The other 5 monocotyledons studied did produce CO₂ in the light. Moreover, the CO₂ compensation point increased linearly with O₂ indicating a stimulation of photorespiration.

The implications of the lack of photorespiration in studies of primary productivity are discussed.

     

C Nuclear Magnetic Resonance Studies of Crassulacean Acid Metabolism in Intact Leaves of Kalanchoë tubiflora

¹³C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoë tubiflora was used to observe Crassulacean acid metabolism in vivo. ¹³C signals from C-4 of malate were observed after overnight exposure of leaves to ¹³CO₂. Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, ¹³C signals were detected in all four carbons of malate in the previously labeled leaves. The ¹³C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.     

Rubisco Oligomers Composed of Linked Small and Large Subunits Assemble in Tobacco Plastids and Have Higher Affinities for CO2 and O2

Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)₈ and (S40L)₁₆ oligomers that are devoid of unlinked S subunits. While there was little or no change in CO₂/O₂ specificity or carboxylation rate of the Rubisco oligomers, their K_ms for CO₂ and O₂ were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called A^NtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in A^NtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO₂-assimilation rates in A^NtS40L at varying pCO₂ corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.