The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles

The influence of indoleacetic acid, 0.03% CO₂, and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO₂ stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO₂. Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO₂, and that indoleacetic acid increased the incorporation of ¹⁴C-bicarbonate into protein. These data are discussed in the context of CO₂-stimulated growth of etiolated tissue, and proposals that CO₂-stimulated growth involves dark CO₂ fixation.     

Photobiology of phytochrome-mediated growth responses in sections of stem tissue from etiolated oats and corn

The far-red reversibility of the phytochrome-controlled stimulation of elongation of coleoptile sections by low fluence red light has been characterized in subapical coleoptile sections from dark-grown Avena sativa L., cv Lodi seedlings. The fluence dependence of the far-red reversal was the same whether or not the very low fluence response is also expressed. The capacity of far-red light to reverse the red light-induced response began to decline if the far-red light was given more than 90 minutes after the red irradiation. Escape was complete if the far red irradiation was given more than 240 minutes after the red irradiation. Sections consisting of both mesocotyl and coleoptile tissue from dark-grown Avena seedlings were found to have physiological regulation of the very low fluence response by indole 3-acetic acid and low external pH similar to that seen for sections consisting entirely of coleoptile tissue. The fluence-dependence of the red light-induced inhibition of mesocotyl elongation was studied in mesocotyl sections from dark grown Zea mays L. hybrid T-929 seedlings. Ten micromolar indole 3-acetic acid stimulates the control elongation of the sections, while at the same time increasing the sensitivity of the tissue for the light-induced inhibition of growth by a factor of 100.     

Enhancement of wall loosening and elongation by Acid solutions

The ability of low pH and CO₂ to induce rapid cell elongation and wall loosening in the Avena coleoptile has been examined with the use of a continuous growth-recording technique and an Instron extensometer, respectively. In particular, the properties of the response to hydrogen ions have been examined in detail and have been compared with the responses initiated by CO₂ and auxin. The optimal pH for growth is about 3.0, and both the maximal growth rate and wall extensibility are similar to that produced by optimal auxin. The timing (initiated in less than 1 minute) and duration (up to 2 hours) of the response to hydrogen ions, as well as certain other aspects of the growth and wall-loosening responses, are described. It is shown that the pH response can be clearly separated from the CO₂ response. Possible mechanisms for the initiation of the growth response to low pH are briefly discussed.     

Bound indoleacetic Acid in Avena coleoptiles

When C¹⁴ carboxyl indoleacetic acid (IAA) is transported through Avena coleoptile sections a fraction of the activity becomes bound. The nature of this bound IAA has been investigated. Upon extraction with solvents and chromatography a substance having the R_F of IAA in 4 solvents was detected. No evidence could be found for the formation of indoleacetyl conjugates. In pea stem sections subjected to a similar experimental regime good evidence was obtained for the occurrence of conjugates. When IAA was supplied exogenously to coleoptile sections floating in solutions the occurrence of conjugates was shown to be dependent on the presence of the primary leaf. In its absence no conjugates could be detected.

On grinding coleoptile sections and subsequent centrifugation at 240 × g the radioactivity was found to be in the tissue fraction as opposed to the supernatant. The radioactivity cannot be removed from the tissue by extraction with water, buffer solution or treatment with ribonuclease. It is readily removed by 10% urea, crystalline trypsin and chymotrypsin. It is therefore concluded that IAA becomes bound to a protein. Bound IAA does not appear to be able to cause growth in Avena coleoptile sections.

     

Enhancement of CO(2) Uptake in Avena Coleoptiles by Fusicoccin

When Avena coleoptile segments are immersed in a solution containing H¹⁴CO₃⁻, the appearance of label in the tissue is stimulated approximately 3-fold by fusicoccin application. This effect is rapid (1-2 minutes lag time), dependent upon respiratory energy, inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not appreciably altered by cycloheximide treatment. A large percentage of the cellular radioactivity is found in the form of malate. Preliminary experiments indicate that CO₂, as opposed to HCO₃⁻, is the favored species of “CO₂” taken up by the segments. These results are consistent with the notion that CO₂, presumably by virtue of its fixation and conversion to malic acid, participates in the early events associated with fusicoccin-enhanced acidification of the cell wall region leading to the stimulation of cell extension growth.     

Regulation of growth in rice seedlings

Etiolated rice seedlings (Oryza sativa L.) exhibited marked morphological differences when grown in sealed containers or in containers through which air was passed continuously. Enhancement of coleoptile and mesocotyl growth and inhibition of leaf and root growth in the sealed containers (“enclosure syndrome”) were accompanied by accumulation of CO₂ and C₂H₄ in and depletion of O₂ from the atmosphere. Ethylene (1 μl 1^?1), high levels of CO₂, and reduced levels of O₂ contributed equally to the increase in coleoptile and mesocotyl growth. The effect of enclosure could be mimicked by passing a gas mixture of 3% O₂, 82% N₂, 15% CO₂ (all v/v), and 1 μl l^?1) C₂H₄ through the vials containing the etiolated seedlings. The effects of high CO₂ and low O₂ concentrations were not mediated through increased C₂H₄ production. The enclosure syndrome was also observed in rice seedlings grown under water either in darkness or in light. The length of the rice coleoptile was positively correlated with the depth of planting in water-saturated vermiculite. The length of coleoptiles of wheat, barley, and oats was not affected by the depth of planting. In rice, the length of coleoptile was determined by the levels of O₂, CO₂, and ethylene, rather than by light. This regulatory mechanism allows rice seedlings to grow out of shallow water in which the concentration of O₂ is limiting.     

Ethylene and the growth of rice seedlings

Etiolated whole rice seedlings enclosed in sealed vials produced ethylene at a rate of 0.9 picomole per hour per seedling. When 2-centimeter-long shoots were subdivided into 5-millimeter-long sections, the sections containing the tip of the shoot evolved 37% of the total ethylene with the remaining 63% being produced along a gradient decreasing to the base of the shoot. The tip of the coleoptile also had the highest level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and of the ethylene-forming enzyme activity. Ethylene is one of the factors controlling coleoptile elongation. Decapitation of the seedling reduced ethylene evolution to one-third its original level and inhibited coleoptile growth. In short-term experiments, the growth rate of decapitated seedlings was restored to almost that of intact seedlings by application of ethylene at a concentration of 10 microliters per liter. Apart from ethylene, O₂ also participates in the control of coleoptile growth. When rice seedlings were grown in a gas mixture of N₂ and O₂, the length of the coleoptiles reached a maximum at a concentration of 2.5% O₂. Lower and higher concentrations of O₂ reduced coleoptile growth. The effect of exogenous ethylene on coleoptile growth was also O₂ dependent.     

Effects of Atmospheric CO(2) Enrichment on the Growth and Mineral Nutrition of Quercus alba Seedlings in Nutrient-Poor Soil

One-year-old dormant white oak (Quercus alba L.) seedlings were planted in a nutrient-deficient forest soil and grown for 40 weeks in growth chambers at ambient (362 microliters per liter) or elevated (690 microliters per liter) levels of CO₂. Although all of the seedlings became severely N deficient, CO₂ enrichment enhanced growth by 85%, with the greatest enhancement in root systems. The growth enhancement did not increase the total water use per plant, so water-use efficiency was significantly greater in elevated CO₂. Total uptake of N, S, and B was not affected by CO₂, therefore, tissue concentrations of these nutrients were significantly lower in elevated CO₂. An increase in nutrient-use efficiency with respect to N was apparent in that a greater proportion of the limited N pool in the CO₂-enriched plants was in fine roots and leaves. The uptake of other nutrients increased with CO₂ concentration, and P and K uptake increased in proportion to growth. Increased uptake of P by plants in elevated CO₂ may have been a result of greater proliferation of fine roots and associated mycorrhizae and rhizosphere bacteria stimulating P mineralization. The results demonstrate that a growth response to CO₂ enrichment is possible in nutrient-limited systems, and that the mechanisms of response may include either increased nutrient supply or decreased physiological demand.     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

Environmental Parameters Affecting Dark Response of Rice Seedlings (Oryza sativa L.) to Triacontanol

Triacontanol applied to IR-8 rice (Oryza sativa L.) seedlings in nutrient solution caused an increase in dry weight during a 6-hour dark period. This increase was altered by atmospheric CO₂ and O₂ concentrations. The largest growth response occurred from 200 to 350 μliters/liter CO₂ with 5% O₂. The treated seedlings did not fix atmospheric CO₂ in the dark, and the immediate products of photosynthesis were not involved in the dry weight increase. The growth response was characterized by an increase in soluble and insoluble Kjeldahl-N, and soluble carbohydrates. The response curve for dry weight increase was a linear function of log presentation time of triacontanol. The response exhibited an apparent K_dose of 25 minutes in 10 μg/liter triacontanol in the dark and 18 minutes in the light. Concentrations of 50 μg/liter and higher inhibited growth.     

Optimization of red light-induced elongation in Avena coleoptile sections and properties of the phytochrome-mediated growth response*

Abstract Optimal conditions for studying the elongation response to a 1 mmol m^?2, 2-min pulse of red light in subapical coleoptile sections from dark-grown oat (Avena sativa L. ev. Lodi) seedlings have been determined. A technique for obtaining standard-length coleoptile sections without exposing either seedlings or sections to any light has been developed, and is described. The optimal conditions found were: sampling time, 12 h after irradiation; buffer conditions, 5 mol m^?3 potassium phosphate with 5% (w/v) sucrose (pH 5.9). The optima were determined by obtaining the time course for light-induced growth under various conditions. The red light-induced growth response is linear until 12 h after irradiation, when it undergoes an interruption. Optimal incubation conditions were determined by varying the buffer contents systematically and measuring the responses at the optimal lime determined. The results indicate a distinct difference between auxin-induced and light-induced growth responses. Even with variations of basal growth rate and several incubation conditions, the red light-induced elongation appears to be of a constant magnitude, to persist for a constant time period. and to exhibit a constant lag period between irradiation and the onset of response. The use of sections that were produced and handled in complete darkness yielded an unusual response to fusicoccin. A linear, high growth rate in response to I mmol m^?3 FC was observed for more than 12 h, both in the irradiated sections and in the dark controls.     

Photorespiration and Oxygen Inhibition of Photosynthesis in Chlorella pyrenoidosa

The inhibition of photosynthesis by O₂ in air-grown Chlorella pyrenoidosa was investigated using three experimental techniques (artificial leaf, aqueous method, and O₂ electrode) to measure carbon assimilation. CO₂ response curves were determined under different O₂, pH, and temperature conditions. Regardless of the experimental technique and condition, O₂ inhibition was not evident until a concentration of 50% was reached; V_max values were reduced whereas K_m (CO₂) values were unaffected by the increasing O₂ concentration. The response of photosynthesis to O₂ was independent of CO₂ and HCO₃⁻ concentrations as well as temperature. Relative rates of photosynthesis showed a 4 to 5% stimulation in 2% O₂, a 12% inhibition in 50% O₂, and a 24% inhibition in 100% O₂. The inhibition by 50% O₂ was still reversible after 20 minutes exposure whereas 100% O₂ caused irreversible inhibition after only 4 minutes.     

Effect of CO(2) Concentration on Glycine and Serine Formation during Photorespiration

Amount and products of photosynthesis during 10 minutes were measured at different ¹⁴CO₂ concentrations in air. With tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) leaves the percentage of ¹⁴C in glycine plus serine was highest (42%) at 0.005% CO₂, and decreased with increasing CO₂ concentration to 7% of the total at 1% CO₂ in air. However, above 0.03% CO₂ the total amount of ¹⁴C incorporated into the glycine and serine pool was about constant. At 0.005% or 0.03% CO₂ the percentage and amount of ¹⁴C in sucrose was small but increased greatly at higher CO₂ levels as sucrose accumulated as an end product. Relatively similar data were obtained with sugar beet (Beta vulgaris L. cv. US H20) leaves. The results suggest that photorespiration at high CO₂ concentration is not inhibited but that CO₂ loss from it becomes less significant.     

A Photorespiratory Mutant of Chlamydomonas reinhardtii

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO₂ concentration for photoautrophic growth in spite of the apparent induction of a functional CO₂ concentrating mechanism in air-adapted cells. In 2% O₂ the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O₂, photosynthetic O₂ evolution is severely inhibited in the mutant by preillumination in limiting CO₂, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O₂ evolution measurements. Net CO₂ uptake is also inhibited when the cells are exposed to air (21% O₂, 0.035% CO₂, balance N₂) for longer than a few minutes. [¹⁴C]Phosphoglycolate accumulates within 5 minutes of photosynthetic ¹⁴CO₂ fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO₂ requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.     

Time course of auxin stimulations of growth

Measurements of the time course of growth responses of corn coleoptile sections to pulses of auxin (10⁻⁵m indoleacetic acid) establish that the growth rate changes in a regular pattern around the auxin pulse: a latent phase of 12 to 15 minutes is followed by an acceleration of growth rate lasting 15 to 20 minutes, after which a fairly steady rate is maintained. When the auxin source is withdrawn, there is an after-effect of about 15 minutes followed by a decay of growth rate, which reaches 50% decay after a further 15 to 40 minutes. The decay phase appears to be a function of the transport of auxin out of the sections. The 50% decay of growth for single cells is estimated at 30 minutes from the time of withdrawal of an exogenous supply of auxin. The regulation of growth by auxin is rapidly imposed or dissipated as auxin enters and exits, respectively, suggesting a facile association and disassociation of auxin with a growth-limiting site in the cell. It is proposed that the growth-stimulated state is dissipated at once when the transportable auxin has passed out of the cell.     

Combined Effects of CO2 Enrichment and Drought Stress on Growth and Energetic Properties in the Seedlings of a Potential Bioenergy Crop Jatropha curcas

The rapid growth of worldwide energy demands has led to mounting concerns about energy shortages and has promoted the development of biofuels, which are susceptible to climate change. To evaluate the effects of future environmental changes such as CO₂ enrichment and water stress on the growth and biodiesel production of bioenergy plants, we exposed Jatropha curcas to two levels of CO₂ concentration (ambient and elevated) and three watering regimes (well-watered, moderate drought, and severe drought) to study its biomass accumulation and allocation, energy cost-gain properties, and photosynthetic response. Elevated CO₂ enhanced biomass accumulation of J. curcas by 31.5, 25.9, and 14.4 % under well-watered, moderate drought, and severe drought treatments, respectively, indicating that the stimulating effect was greater under optimum water conditions than in water-deficit conditions. Drought stress significantly increased the biomass allocation to roots, especially the fine roots. CO₂ enrichment also increased the root mass fraction, though not significantly. CO₂ enrichment significantly enhanced the photosynthetic rate measured under growth CO₂ concentration (A _growth) and decreased foliar N content and therefore construction cost irrespective of watering conditions. Under elevated CO₂, J. curcas employed a quicker return energy use strategy indicated by the higher photosynthetic energy use efficiency and lower payback time. There was a pronounced downregulation in the light-saturated photosynthetic rate under the common CO₂ concentration (P _max) under long-term CO₂ exposure, due to a decrease in the initial and total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities and partially lower foliar N content. The significant interaction of CO₂ enrichment and watering regimes implied that the stimulation of plant growth by CO₂ enrichment may be negated by soil drought in the future. Long-term field experiments manipulating multiple factors simultaneously are needed to explore how the ecophysiological traits measured for J. curcas translate into bioenergy production.     

Carbonic Anhydrase and the Regulation of Photosynthesis

THE role of CO₂ in the regulation of photosynthetic and respiratory metabolism in plants is little understood in the unicellular alga Chlorella pyrenoidosa; for example, after autotrophic growth in high CO₂ (5·5% by volume), transfer to a CO₂ concentration about ten times less than the concentration in air results initially in low rates of photosynthesis characterized by the virtual absence of the Calvin cycle¹ of CO₂ fixation². An induction period of about 2 h is necessary before normal photosynthetic rates are established. Cells grown in air (0.03% CO₂) do not show this effect and photosynthesize at comparatively high rates even in very low concentrations of CO₂.     

Osmoregulation in the Avena Coleoptile : CONTROL OF SOLUTE UPTAKE IN PEELED SECTIONS

Peeled Avena sativa coleoptile sections (i.e. sections from which the epidermis has been removed) have been used to study the control of solute uptake under conditions where the uptake is not limited by the cuticular barrier. In the presence of 2% sucrose, auxin enhances the rate at which the total osmotic solutes increase, but this appears to be a response to the increased growth rate, inasmuch as the auxin effect is eliminated when growth is inhibited osmotically. When sections are incubated in sucrose or in 20 millimolar NaCl, the osmotic concentration increases until a plateau is reached after 8 to 24 hours. Auxin has no effect on the initial rate of increase in osmotic concentration but causes the osmotic concentration to reach a plateau earlier and at a lower osmotic conentration value. This difference in steady-state osmotic concentration is, in part, a response to auxin itself, as it persists when auxin-induced growth is inhibited osmotically. The upper limit for osmotic concentration does not appear to be determined by the turgor pressure, inasmuch as a combination of sucrose and NaCl gave a higher plateau osmotic concentration than did either solute alone. We suggest that the rate of solute uptake is determined by the availability of absorbable solutes and by the surface area exposed to the solutes. Each absorbable solute reaches a maximum internal concentration independent of other absorbable solutes; the steady-state osmotic concentration is simply the sum of these individual internal concentrations.     

Effect of pH and Auxin on Chloride Uptake into Avena Coleoptile Cells

The effect of pH on ³⁶Cl⁻ movement into coleoptile cells (Avena sativa L. cv. Garry) was investigated and compared with effects of indoleacetic acid. ³⁶Cl⁻ uptake, but not efflux, is stimulated when coleoptile sections are placed in media adjusted to pH levels from 5 to 3 after a preincubation period at pH 6.5. The enhancement is seen within 2 minutes, is not correlated with growth, and is completely erased by respiratory inhibitors. In comparison to the acid-induced stimulation, the stimulatory effect of indoleacetic acid on ³⁶Cl⁻ uptake is also not accompanied by accelerated efflux, and indoleacetic acid does not further stimulate ³⁶Cl⁻ uptake into 1-millimeter sections beyond that seen at pH 3.5 without auxin.     

Enhanced production of biomass and lipids by supplying CO2 in marine microalga Dunaliella sp.

Non-food-based biofuel feedstocks are in high demand worldwide. Among the various feedstocks, microalgae are the most promising feedstock for mitigating atmospheric CO₂ and producing biodiesel. In this study, various concentrations of CO₂, from 0.03 to 12%, were used to investigate their effect on the cell growth, biomass and lipid production and fatty acid composition of Dunaliella sp. in a closed photobioreactor. The results showed that the highest biomass and total lipids, 521 mg/L/d and 40 mg/L/d, respectively, were produced with 5% CO₂ aeration during the logarithmic growth phase. The oleic acid (18:1n9c) and elaidic acid (18:1n9t) contents were increased approximately two fold. The physiological responses of Dunaliella sp. at 10% CO₂ were similar to those at 5% CO₂. Therefore, the present results suggest that 5–10% is a suitable CO₂ concentration range for Dunaliella sp. growth to mitigate atmospheric CO₂ and increase biofuel production.