Changing Responses of Stomata to Abscisic Acid and CO2 as Leaves and Plants Age

Willmer, C. M., Wilson, A. B. and Jones, H. G. 1988. Changingresponses of stomata to abscisic acid and CO₂ as leaves andplants age.—J. exp. Bot. 39: 401–410. Stomatal conductances were measured in ageing leaves of Commelinacommunis L. as plants developed; stomatal responses to CO₂ andabscisic acid (ABA) in epidermal strips of C. communis takenfrom ageing leaves of developing plants and in epidermal stripsfrom the same-aged leaves (the first fully-expanded leaf) ofdeveloping plants were also monitored. Stomatal behaviour wascorrelated with parallel measurements of photosynthesis andleaf ABA concentrations. Stomatal conductance in intact leavesdecreased from a maximum of 0-9 cm s^{– 1} at full leaf expansionto zero about 30 d later when leaves were very senescent. Conductancesdeclined more slowly with age in unshaded leaves. Photosynthesisof leaf slices also declined with age from a maximum at fullleaf expansion until about 30 d later when no O₂ exchange wasdetectable. Exogenously applied ABA (0.1 mol m^{– 3}) didnot affect respiration or photosynthesis. In epidermal stripstaken from ageing leaves the widest stomatal apertures occurredabout 10 d after full leaf expansion (just before floweringbegan) and then decreased with age; this decrease was less dramaticin unshaded leaves. The inhibitory effects of ABA on stomatalopening in epidermal strips decreased as leaves aged and wasgreater in the presence of CO₂ than in its absence. When leaveswere almost fully-senescent stomata were still able to open.At this stage, guard cells remained healthy-looking with greenchloroplasts while mesophyll cells were senescing and theirchloroplasts were yellow. Similar data were obtained for stomatain epidermal strips taken from the same-aged leaves of ageingplants. The inhibitory effects of ABA on stomatal opening alsodecreased with plant age. In ageing leaves both free and conjugated ABA concentrationsremained low before increasing dramatically about 30 d afterfull leaf expansion when senescence was well advanced. Concentrationsof free and conjugated ABA remained similar to each other atall times. It is concluded that the restriction of stomatal movements inintact leaves as the leaves and plants age is due mainly toa fall in photosynthetic capacity of the leaves which affectsintracellular CO₂ levels rather than to an inherent inabilityof the stomata to function normally. Since stomatal aperturein epidermal strips declines with plant and leaf age and stomatabecome less responsive to ABA (while endogenous leaf ABA levelsremain fairly constant until leaf senescence) it is suggestedthat some signal, other than ABA, is transmitted from the leafor other parts of the plant to the stomata and influences theirbehaviour. Key words: Abscisic acid, CO₂, Commelina, leaf age, senescence, stomatal sensitivity     

Localization of Carbonic Anhydrase in Mesophyll Cells of Terrestrial C3 Plants in Relation to CO2 Assimilation

The activity and intracellular compartmentation of carbonicanhydrase was examined in mesophyll protoplasts of several C₃terrestrial species including wheat, since this enzyme may facilitatediffusion of inorganic carbon in solution by converting CO₂to bicarbonate. Carbonic anhydrase was located in the mesophyllchloroplast with little or no activity in the cytosolic fraction.In wheat, carbonic anhydrase was absent in etiolated leavesand increased in the light during greening. Thus the enzymemay have a role in photosynthesis in the chloroplast but notin the cytosol of mesophyll cells of higher C₃ plants. The amount of CO₂ required for half maximum rates of photosynthesis(under low O₂) was about two-fold higher for isolated protoplaststhan with isolated chloroplasts of wheat. The form of inorganiccarbon taken up by protoplasts, like that of chloroplasts, isCO₂. The results are discussed in relation to a possible resistanceto CO₂ transfer in the cytosol of mesophyll cells. (Received February 25, 1985; Accepted May 7, 1985)     

Physiological and Growth Responses of C3 and C4 Plants to Reduced Temperature When Grown at Low CO2 of the Last Ice Age

During the last Ice age, CO2 concentration ([CO2]) was 180-200 μmol/mol compared with the modern value of 380 μmol/mol,and global temperatures were ～8 ℃ cooler. Relatively little is known about the responses of C3 and C4 species to longterm exposure to glacial conditions. Here Abutilon theophrasti Medik. (C3) and Amaranthus retroflexus L. (C4) were grown at 200 μmol/mol CO2 with current (30/24 ℃) and glacial (22/16 ℃) temperatures for 22 d. Overall, the C4 species exhibited a large growth advantage over the C3 species at low [CO2]. However, this advantage was reduced at low temperature, where the C4 species produced 5× the total mass of the C3 species versus 14× at the high temperature.This difference was due to a reduction In C4 growth at low temperature, since the C3 species exhibited similar growth between temperatures. Physiological differences between temperatures were not detected for either species, although photorespirationlnet photosynthesis was reduced in the C3 species grown at low temperature, suggesting evidence of improved carbon balance at this treatment. This system suggests that C4 species had a growth advantage over C3 species during low [CO2] of the last ice age, although concurrent reductions in temperatures may have reduced this advantage.     

Humidity Responses of Stomata and the Potassium Content of Guard Cells

Humidity responses of stomata and changes in the potassium contentof their guard cells were investigated in intact plants anddetached epidermal strips of Valerianella locusta (L.) Betcke.Potassium content was determined by Macallum‘s stain.It was found that changes in stomatal aperture caused by decreasingor increasing humidity were followed only after a delay by changesin the potassium content of the guard cells. By comparison,if stomatal movements occurred in response to changes in illuminationthe relative potassium content of the guard cells correlatedcontinuously with the changes in stomatal aperture. Since thepotassium content of the guard cells changed only after mostof the stomatal movements in response to changes in humiditywere completed changes in potassium content and humidity responsesof stomata can be described as following a hysteresis curve.     

Responses of C4 grasses to atmospheric CO2 enrichment

Summary The growth and photosynethetic responses to atmospheric CO₂ enrichment of 4 species of C₄ grasses grown at two levels of irradiance were studied. We sought to determine whether CO₂ enrichment would yield proportionally greater growth enhancement in the C₄ grasses when they were grown at low irradiance than when grown at high irradiance. The species studied were Echinochloa crusgalli, Digitaria sanguinalis, Eleusine indica, and Setaria faberi. Plants were grown in controlled environment chambers at 350, 675 and 1,000 l 1^-1 CO₂ and 1,000 or 150 mol m^-2 s^-1 photosynthetic photon flux density (PPFD). An increase in CO₂ concentration and PPFD significantly affected net photosynthesis and total biomass production of all plants. Plants grown at low PPFD had significantly lower rates of photosynthesis, produced less biomass, and had reduced responses to increases in CO₂. Plants grown in CO₂-enriched atmosphere had lower photosynthetic capacity relative to the low CO₂ grown plants when exposed to lower CO₂ concentration at the time of measurement, but had greater rate of photosynthesis when exposed to increasing PPFD. The light level under which the plants were growing did not influence the CO₂ compensation point for photosynthesis.     

Temperature Dependence of the Linkage of Quantum Yield of Photosystem II to CO2 Fixation in C4 and C3 Plants

The temperature dependence of quantum yields of electron transport from photosystem II (PSII) ([phi]II, determined from chlorophyll a fluorescence) and CO2 assimilation ([phi]CO2, apparent quantum yield for CO2 assimilation) were determined simultaneously in vivo. With C4 species representing NADP-malic enzyme, NAD-malic enzyme, and phosphoenolpyruvate carboxykinase subgroups, the ratio of [phi]II/[phi]CO2 was constant over the temperature range from 15 to 40[deg]C at high light intensity (1100 [mu]mol quanta m-2 s-1). A similar response was obtained at low light intensity (300 [mu]mol quanta m-2 s-1), except the ratio of [phi]II/[phi]CO2 increased at high temperature. When the true quantum yield for CO2 fixation ([phi]CO2*) was calculated by correcting for respiration in the light (estimated from temperature dependence of dark respiration), the ratio of [phi]II/[phi]C02* remained constant with varying temperature and under both light intensities in all C4 species examined. Because the [phi]II/[phi]CO2* ratio was the same in C4 monocots representing the three subgroups, the ratio was not affected by differences in the bio-chemical mechanism of concentrating CO2 in the bundle sheath cells. The results suggest that PSII activity is closely linked to the true rate of CO2 fixation in C4 plants. The close relationship between [phi]II and [phi]CO2* in C4 species under varying temperature and light intensity conditions is apparently due to a common low level of photorespiration and a primary requirement for reductive power in the C3 pathway. In contrast, in a C3 plant the [phi] II/[phi]CO2* ratio is higher under normal atmospheric conditions than under nonphotorespiratory conditions and it increases with rising temperature. This decrease in efficiency in utilizing energy derived from PSII for CO2 fixation is due to an increase in photorespiration. In both the C3 and C4 species, photochemistry is limited under low temperature, and thus excess energy must be dissipated by nonphotochemical means.     

The Responses of Stomatal Density to CO2 Partial Pressure

Experiments on a range of species of tree, shrub and herb haveshown that stomatal density and stomatal index increase as thepartial pressure of CO₂ decreases over the range from the currentlevel of 34 Pa to 22.5 Pa. Stomatal density responds to thereduced partial pressure of CO₂ in a simulation of high altitude(3000 m), when the CO₂ mole fraction is unchanged. When the partial pressure of CO₂ is increased from 35 to 70Pa stomatal density decreases slightly, with a response to unitchange in CO₂ which is about 10% of that below 34 Pa. Measurements of gas exchange on leaves which had developed indifferent CO₂ partial pressures, but at low saturation vapourpressure deficits in the range of 0.7 to 0.9 kPa, indicatedlower photosynthetic rates but higher stomatal conductancesat reduced CO₂ partial pressures. Experiments on populations of Nardus stricta originating fromaltitudes of 366 m and 810 m in Scotland, indicated geneticdifferences in the responses of stomatal density to CO₂ in pressuressimulating altitudes of sea level and 2 000 m. Plants from thehigher altitude showed greater declines in stomatal densitywhen the CO₂ partial pressure was increased. Key words: Stomata, CO₂, gas exchange, altitude, atmospheric pressure     

The Structure and Orientation of Guard Cells in Plants Showing Stomatal Responses to Changing Vapour Pressure Difference

Transmission electron micrographs revealed that a substantialpart of the guard cell wall of both Quercus robur L. and Populusnigra ‘italica’ L. was either free of cuticle orcovered with a greatly reduced cuticular layer. In Quercus thestructure of the guard cell was such that the area of limitedcuticular development would be exposed to the evaporating powerof the atmosphere even when the stomata were closed. Lanthanumstaining confirmed that this area might be an important siteof evaporation. A similar evaporation site was identified inthe guard cell wall of Pinus sylvestris L. Light micrographsrevealed that this area could also be exposed on the outsideof the leaf when the stomata were closed. It appears that guardcell orientation with respect to the epidermal plane dependsupon epidermal turgor. Changes in orientation of the guard cellcoupled with the exact location of the cuticle-free area inthe guard cell wall may explain the nature of the stomatal responseof individual species to changing VPD and the effect of othervariables, e.g. water deficit, on this response. Quercus robur L, oak, Populus nigra L, poplar, stomata, guard cells, cuticle, evaporation, vapour pressure difference     

CO2 Assimilation and Partitioning of Carbon in Maize Plants Deprived of Orthophosphate

In order to study the effects of inorganic phosphate (P1) starvationon C₄plants, 3-week-old maize plants (Zea maysL cv. Brulouis)were grown in a growth chamber on a nutrient solution withoutP1 over 22 d During the first 2 weeks, Pi-starved plants grewas well as control plants The Pi concentration in the planttissue decreased rapidly with time, which suggests that normalbiomass production can be maintained at the expense of internalP1 In addition, photosynthetic CO2 assimilation measured 4-6h after dawn was not affected, but the concentration of glucose,sucrose, and starch in leaves was much higher than in the controls¹⁴CO₂ pulse-chase experiments earned out on the ninth day oftreatment showed that ¹⁴CO₂ assimilation was perturbed duringthis initial period, resulting in a larger flow of carbon toboth starch and sucrose At the beginning of the third week ofP1 starvation (15 d after treatment) ¹⁴C incorporation intosucrose stayed high relative to controls but this was not thecase for starch At the end of the third week of P1-deficiency,shoot growth was considerably reduced and fresh weight was onlyone-third of that of the control plants. The P1 concentrationof both the leaf and root tissues was less than 1.0 µmolg^–1 FW compared to 20-25µmol g¹ FW in the controls.Photosynthetic CO₂ assimilation was reduced and the leaf concentrationof sucrose and starch, which had begun to decrease after theend of the second week of P1 limitation, became lower than inthe controls. These results obtained on maize plants show thatphotosynthesis and carbon partitioning between sucrose and starchwere strongly affected by P1 deficiency, similar to C₃ species. Key words: CO₂ assimilation, corn, orthophosphate deficiency, starch, sucrose     

Modification of the CO2 Responses of Maize Stomata by Abscisic Acid and by Naturally-Occurring and Synthetic Cytokinins

Fragments of maize leaves were incubated at controlled temperatureand irradiance either on distilled water or on one of threeconcentrations of cytokinin (10^–1, 10^–2 and 10^–3mol m^–3). The effects of zeatin or kinetin on stomatalaperture were determined by stripping abaxial epidermis fromthe fragments after incubation and immediately measuring stomatalapertures under the microscope. At each cytokinin concentrationleaf pieces were incubated at 5 or 350 µmol mol^–1CO₂ with or without ABA (10^–1 mol m^–3). At 5.0 µmolmol^–1 CO₂ increasing the concentrations of zeatin hada negligible effect upon stomatal aperture. When air containing350 umol mol^–1 CO₂ was bubbled through the incubationsolutions, apertures of stomata incubated on water were morethan halved. Increasing cytokinin concentrations reduced theeffect of CO₂ on stomata and incubation on 10^–1 mol m^–3zeatin completely removed any CO₂ response. The addition ofABA restored the effect of CO₂, even at the highest cytokininconcentration. Key words: Maize, CO₂ response, ABA, Cytokinins     

Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants

Rates of CO₂ assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO₂ in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25°C. Electron transport activity for CO₂ assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O₂, and between fluorescence and rates of CO₂ assimilation at 19 millibars but not 200 millibars O₂. The values of fluorescence measured at about 220 microbars intercellular CO₂ were not greatly affected by increasing O₂ from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO₂ partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO₂ partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO₂ partial pressures which reversed to a positive correlation at higher values of CO₂ partial pressures. Similar results were observed in the C₃ species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C₄ species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO₂ assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q_E) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed.     

Relationship Between Photosystem 2 Electron Transport and Photosynthetic CO2 Assimilation Responses to Irradiance in Young Apple Tree Leaves

The responses to irradiance of photosynthetic CO₂ assimilation and photosystem 2 (PS2) electron transport were simultaneously studied by gas exchange and chlorophyll (Chl) fluorescence measurement in two-year-old apple tree leaves (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd). Net photosynthetic rate (P _N) was saturated at photosynthetic photon flux density (PPFD) 600-1 100 (mol m^-2 s^-1, while the PS2 non-cyclic electron transport (P-rate) showed a maximum at PPFD 800 mol m^-2 s^-1. With PPFD increasing, either leaf potential photosynthetic CO₂ assimilation activity (F_d/F_s) and PS2 maximal photochemical activity (F_v/F_m) decreased or the ratio of the inactive PS2 reaction centres (RC) [(F_i – F_o)/(F_m – F_o)] and the slow relaxing non-photochemical Chl fluorescence quenching (q_s) increased from PPFD 1 200 mol m^-2 s^-1, but cyclic electron transport around photosystem 1 (RFp), irradiance induced PS2 RC closure [(F_s – F_o)/F_m – F_o)], and the fast and medium relaxing non-photochemical Chl fluorescence quenching (q_f and q_m) increased remarkably from PPFD 900 (mol m^-2 s^-1. Hence leaf photosynthesis of young apple leaves saturated at PPFD 800 mol m^-2 s^-1 and photoinhibition occurred above PPFD 900 mol m^-2 s^-1. During the photoinhibition at different irradiances, young apple tree leaves could dissipate excess photons mainly by energy quenching and state transition mechanisms at PPFD 900-1 100 mol m^-2 s^-1, but photosynthetic apparatus damage was unavoidable from PPFD 1 200 mol m^-2 s^-1. We propose that Chl fluorescence parameter P-rate is superior to the gas exchange parameter P _N and the Chl fluorescence parameter F_v/F_m as a definition of saturation irradiance and photoinhibition of plant leaves.     

Maximum Quantum Yields of O2 Evolution in C4 Plants under High CO2

The quantum yields of photosynthetic O₂ evolution were measuredin 15 species of C₄ plants belonging to three different decarboxylationtypes (NADP-ME type, NAD-ME type and PEP-CK type) and 5 speciesof C₃ plants and evaluated relative to the maximum theoreticalvalue of 0.125 mol oxygen quanta^-1. At 25°C and 1% CO₂,the quantum yield in C₄ plants averaged 0.079 (differences betweensubgroups not significant) which was significantly lower thanthe quantum yield in C₃ plants (average of 0.105 for 5 species).This lower quantum yield in C₄ plants is thought to reflectthe requirement of energy in the C₄ cycle. For the C₄ NADP-MEtype plant Z. mays and NAD-ME type plant P. miliaceum, quantumyields were also measured over a range of CO₂ levels between1 and 20%. In both species maximum quantum yields were obtainedunder 10% CO₂ (0.105 O₂ quanta_-1 in Z. mays and 0.097 O₂ quanta_-1in P. miliaceum) indicating that at this CO₂ concentration thequantum yields are similar to those obtained in C₃ plants underCO₂ saturation. The high quantum yield values in C₄ plants undervery high CO₂ may be accomplished by direct diffusion of atmosphericCO₂ to bundle sheath cells, its fixation in the C₃ pathway,and feedback inhibition of the C₄ cycle by inorganic carbon. (Received June 6, 1995; Accepted August 15, 1995)     

Variation in Quantum Yield for CO(2) Uptake among C(3) and C(4) Plants

The quantum yield for CO₂ uptake was measured on a number of C₃ and C₄ monocot and dicot species. Under normal atmospheric conditions (330 microliters per liter CO₂, 21% O₂) and a leaf temperature of 30°C, the average quantum yields (moles CO₂ per einstein) were as follows: 0.052 for C₃ dicots, 0.053 for C₃ grasses, 0.053 for NAD-malic enzyme type C₄ dicots, 0.060 for NAD-malic enzyme type C₄ grasses, 0.064 for phosphoenolpyruvate carboxykinase type C₄ grasses, 0.061 for NADP-malic enzyme C₄ dicots, and 0.065 for NADP-malic enzyme type C₄ grasses. The quantum yield under normal atmospheric conditions was temperature dependent in C₃ species, but apparently not in C₄ species. Light and temperature conditions during growth appeared not to influence quantum yield. The significance of variation in the quantum yields of C₄ plants was discussed in terms of CO₂ leakage from the bundle sheath cells and suberization of apoplastic regions of the bundle sheath cells.     

Control of the Co2 Responses of Stomata by Indol-3ylacetic Acid and Abscisic Acid

Previous studies of stomatal behaviour on detached epidermisof Commelina communis L. have suggested that abscisic acid (ABA)and C02 act independently to cause stomatal closure. Evidenceis presented here that if indol-3ylacetic acid (IAA) is addedto the medium used for incubating the epidermis, an interactionbetween ABA and Co₂ becomes apparent. Increasing concentrationsof IAA reduce the ability of the stomata to respond to CO₂,and ABA appears to antagonize this effect. Recognition of therole of IAA enables us to reconcile earlier conflicting reportsconcerning the interdependence of effects of ABA and Co₂on stomata.     

Delay in the Response of Stomata to Abscisic Acid in CO2-free Air

Abscisic acid (10^–5 M) was fed via their petioles to leavesdetached from well watered plants of Xanthium strumartum, whilethe intercellular spaces were flushed with air of known CO₂content. A closing response to ABA occurred in the presenceor absence of CO₂, and the stomata responded to CO₂ whetheror not ABA was supplied to the leaves. A factorial experimentrevealed no interaction between CO₂ and ABA, and suggested thattheir effect on the rate of closure was purely additive. Theonly evidence of interdependence between the two corn poundswas a delay in the response to ABA in C0 air, which was moremarked in a high light intensity. A hypothesis which is consistentwith the data is that ABA induces stomatal closure by interferingwith the energy supply required for the active transport processeson which guard cell turgor depends. The inhibitory action ofABA takes longer in CO₂-free air because, in the absence ofCO₂ fixation, energy is available from chioroplasts as wellas mitochondria.     

Short-Term and Long-Term Responses of Crassulacean Acid Metabolism Plants to Elevated CO(2)

For the leaf succulent Agave deserti and the stem succulent Ferocactus acanthodes, increasing the ambient CO₂ level from 350 microliters per liter to 650 microliters per liter immediately increased daytime net CO₂ uptake about 30% while leaving nighttime net CO₂ uptake of these Crassulacean acid metabolism (CAM) plants approximately unchanged. A similar enhancement of about 30% was found in dry weight gain over 1 year when the plants were grown at 650 microliters CO₂ per liter compared with 350 microliters per liter. Based on these results plus those at 500 microliters per liter, net CO₂ uptake over 24-hour periods and dry weight productivity of these two CAM succulents is predicted to increase an average of about 1% for each 10 microliters per liter rise in ambient CO₂ level up to 650 microliters per liter.     

C3和C4植物的氮素利用机制

提高植物的氮素利用效率(NUE)不仅有利于保障全球粮食安全, 也是实现农业可持续发展的重要途径。近半个世纪以来, 植物氮素利用机理研究已取得重要进展, 但NUE的调控机制仍不明确, NUE的提高仍然十分有限。高等植物集光合碳素同化和氮素同化于一体, 只有碳氮代谢相互协调, 才能维持植物体内的碳氮平衡, 保证植物正常生长发育。由于C₃和C₄植物的光合氮素利用率(PNUE)存在差异, 对氮素的利用效率也会存在差异。为了更有效地提高作物的NUE, 须更全面地了解C₃和C₄植物对氮素吸收、转运、同化和信号转导等关键因子的功能和调控机制。此外, 面对大气CO₂浓度增高和全球气候变暖条件下的植物碳氮同化及其机理的研究也不容忽视。该文综述了C₃和C₄植物氮素利用关键因素的差异及其调控机制, 并对提高C₃禾本科作物氮素利用效率的遗传改良途径进行了展望。     

C3、C4和C3-C4中间型植物的进化

介绍了有关C3、C4和C3-C4中间型植物进化的形态学、生理学、分子生物学、遗传学等方面的证据;推断地球上首先出现C3植物,然后是C3-C4中间类型植物,最后出现C4植物.