Asymmetric responses of adaxial and abaxial stomata to elevated CO2: impacts on the control of gas exchange by leaves

The response of adaxial and abaxial stomatal conductance in Rumex obtusifolius to growth at elevated atmospheric concentrations of CO₂ (250 μmol mol^?1 above ambient) was investigated over two growing seasons. The conductance of both the adaxial and abaxial leaf surfaces was found to be reduced by elevated concentrations of CO₂. Elevated CO₂ caused a much greater reduction in conductance for the adaxial surface than for the abaxial surface. The absence of effects upon stomatal density indicated that the reductions were probably the result of changes in stomatal aperture. Partitioning of gas exchange between the leaf surfaces revealed that increased concentrations of CO₂ caused increased rates of photosynthesis only via the abaxial surface. Additionally, leaf thickness was found to increase during growth at elevated concentrations of CO₂. The tendency for these amphistomatous leaves to develop a distribution of conductance approaching that of hypostomatous leaves clearly reduced their maximum photosynthetic potential. This conclusion was supported by measurements of stomatal limitation, which showed greater values for the adaxial surfaces, and greater values at elevated CO₂. This reduction in photosynthesis may in part be caused by higher diffusive limitations imposed because of increased leaf thickness. In an uncoupled canopy, asymmetrical stomatal responses of the kind identified here may appreciably reduce transpiration. Species which show symmetrical responses are less likely to show reduced transpirational rates, and a redistribution of water loss between species may occur. The implications of asymmetrical stomatal responses for photosynthesis and canopy transpiration are discussed.     

Photosynthetic and structural acclimation to light direction in vertical leaves of Silphium terebinthinaceum

The azimuth of vertical leaves of Silphium terebinthinaceum profoundly influenced total daily irradiance as well as the proportion of direct versus diffuse light incident on the adaxial and abaxial leaf surface. These differences caused structural and physiological adjustments in leaves that affected photosynthetic performance. Leaves with the adaxial surface facing East received equal daily integrated irradiance on each surface, and these leaves had similar photosynthetic rates when irradiated on either the adaxial or abaxial surface. The adaxial surface of East-facing leaves was also the only surface to receive more direct than diffuse irradiance and this was the only leaf side which had a clearly defined columnar palisade layer. A potential cost of constructing East-facing leaves with symmetrical photosynthetic capcity was a 25% higher specific leaf mass and increased leaf thickness in comparison to asymmetrical South-facing leaves. The adaxial surface of South-facing leaves received approximately three times more daily integrated irradiance than the abaxial surface. When measured at saturating CO₂ and irradiance, these leaves had 42% higher photosynthetic rates when irradiated on the adaxial surface than when irradiated on the abaxial surface. However, there was no difference in photosynthesis for these leaves when irradiated on either surface when measurements were made at ambient CO₂. Stomatal distribution (mean adaxial/abaxial stomatal density = 0.61) was unaffected by leaf orientation. Thus, the potential for high photosynthetic rates of adaxial palisade cells in South-facing leaves at ambient CO₂ concentrations may have been constrained by stomatal limitations to gas exchange. The distribution of soluble protein and chlorophyll within leaves suggests that palisade and spongy mesophyll cells acclimated to their local light environment. The protein/chlorophyll ratio was high in the palisade layers and decreased in the spongy mesophyll cells, presumably corresponding to the attentuation of light as it penetrates leaves. Unlike some species, the chlorophyll a/b ratio and the degree of thylakoid stacking was uniform throughout the thickness of the leaf. It appears that sun-shade acclimation among cell layers of Silphium terebinthinaceum leaves is accomplished without adjustment to the chlorophyll a/b ratio or to thylakoid membrane structure.     

On the relationship between leaf anatomy and CO2 diffusion through the mesophyll of hypostomatous leaves

Internal conductances to CO₂ transfer from the stomatal cavity to sites of carboxylation (g_i) in hypostomatous sun-and shade-grown leaves of citrus, peach and Macadamia trees (Lloyd et al. 1992) were related to anatomical characteristics of mesophyll tissues. There was a consistent relationship between absorptance of photosynthetically active radiation and chlorophyll concentration (mmol m^?2) for all leaves, including sclerophyllous Macadamia, whose transmittance was high despite its relatively thick leaves. In thin peach leaves, which had high g_i, the chloro-plast volume and mesophyll surface area exposed to intercellular air spaces (ias) per unit leaf area were similar to those in the thicker leaves of the evergreen species. Peach leaves, however, had the lowest leaf dry weight per area (D/a), the lowest tissue density (T_d) and the highest chloro-plast surface area (S_c) exposed to ias. There were negative correlations between g_i and leaf thickness or D/a, but positive correlations between g_i and S_c or S_c/T_d. We developed a one-dimensional diffusion model which partitioned g_i into a gaseous diffusion conductance through the ias (g_ias) plus a liquid-phase conductance through mesophyll cell walls (g_cw). The model accounted for a significant amount of variation (r₂=0.80) in measured g_i by incorporating both components. The g_ias component was related to the one-dimensional path-length for diffusion across the mesophyll and so was greater in thinner peach leaves than in leaves of evergreen species. The g_cw component was related to tissue density and to the degree of chloroplast exposure to the ias. Thus the negative correlations between g_i and leaf thickness or D/a related to g_ias whereas positive correlations between g_i and S_c or S_c/T_d, related to g_cw. The g_cw was consistently lower than g_ias, and thus represented a greater constraint on CO₂ diffusion in the mesophylls of these hypostomatous species.     

Temperature response of carbon isotope discrimination and mesophyll conductance in tobacco

The partial pressure of CO₂ at the sites of carboxylation within chloroplasts depends on the conductance to CO₂ diffusion from intercellular airspace to the sites of carboxylation, termed mesophyll conductance (g_m). We investigated the temperature response of g_m in tobacco (Nicotiana tabacum) by combining gas exchange in high light, ambient CO₂ in either 2 or 21% O₂ with carbon isotope measurements using tuneable diode laser spectroscopy. The g_m increased linearly with temperature in 2 or 21% O₂. In 21% O₂, isotope discrimination associated with g_m decreased from 5.0 ± 0.2 to 1.8 ± 0.2‰ as temperature increased from 15 to 40 °C, but the photorespiratory contribution to the isotopic signal is significant. While the fractionation factor for photorespiration (f = 16.2 ± 0.7‰) was independent of temperature between 20 and 35 °C, discrimination associated with photorespiration increased from 1.1 ± 0.01 to 2.7 ± 0.02‰ from 15 to 40 °C. Other mitochondrial respiration contributed around 0.2 ± 0.03‰. The drawdown in CO₂ partial pressure from ambient air to intercellular airspaces was nearly independent of leaf temperature. By contrast, the increase in g_m with increasing leaf temperature resulted in the drawdown in CO₂ partial pressure between intercellular airspaces and the sites of carboxylation decreasing substantially at high temperature.     

Inclination of sun and shade leaves influences chloroplast light harvesting and utilization

Light harvesting and utilization by chloroplasts located near the adaxial vs the abaxial surface of sun and shade leaves were examined by fluorometry in two herbaceous perennials that differed in their anatomy and leaf inclination. Leaves of Thermopsis montana had well-developed palisade and spongy mesophyll whereas the photosynthetic tissue of Smilacina stellata consisted of spongy mesophyll only. Leaf orientation depended upon the irradiance during leaf development. When grown under low-light levels, leaves of S. stellata and T. montana were nearly horizontal, whereas under high-light levels, S. stellata leaves and T. montana leaves were inclined 60⁰ and 30⁰, respectively. Leaf inclination increased the amount of light that was intercepted by the lower leaf surfaces and affected the photosynthetic properties of the chloroplasts located near the abaxial leaf surface. The slowest rates of quinone pool reduction and reoxidation were found in chloroplasts located near the adaxial leaf surface of T. montana plants grown under high light, indicating large quinone pools in these chloroplasts. Chloroplasts near the abaxial surface of low-light leaves had lower light utilization capacities as shown by photochemical quenching measurements. The amount of photosystem II (PSII) down regulation, measured from each leaf surface, was also found to be influenced by irradiance and leaf inclination. The greatest difference between down regulation monitored from the adaxial vs abaxial surfaces was found in plants with horizontal leaves. Different energy dissipation mechanisms may be employed by the two species. Values for down regulation in S. stellata were 2–3 times higher than those in T. montana, while the portion of the PSII population which was found to be Q_B nonreducing was 4–6 times lower in high light S. stellata leaves than in T. montana. All values of Stern-Volmer type nonphotochemical quenching (NPQ) from S. stellata leaves were similar when quenching analysis was performed at actinic irradiances that were higher than the irradiance to which the leaf surface was exposed during growth. In contrast, with T. montana, NPQ values from the abaxial leaf surface were up to 45% higher than those from the adaxial leaf surface regardless of growth conditions. The observed differences in chloroplast properties between species and between the adaxial and abaxial leaf surfaces may depend upon a complex interaction among light, leaf anatomy and leaf inclination.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Leaf bisection for the enzymatic isolation of mesophyll protoplasts fromSaintpaulia ionantha

We describe a rapid and efficient procedure for isolating leaf mesophyll protoplasts fromSaintpaulia ionantha (African violet). Because of the unusual surface properties of the leaf, efficient digestion required bisection of the lamina into abaxial and adaxial halves to expose the mesophyll. Enzymatic digestion occurred during a 3 h incubation with 2% (m/v) driselase in 0.50 M mannitol, yielding 2–3 × 10⁵ protoplasts g⁻¹ (f.m.), with 70% viability. Protoplasts ranged in size from 50 to 100μm, and were derived from mostly non-photosynthetic mesophyll parenchyma.     

Photosystem II efficiency of the palisade and spongy mesophyll in <Emphasis Type="Italic">Quercus coccifera</Emphasis> using adaxial/abaxial illumination and excitation light sources with wavelengths varying in penetration into the leaf tissue

The existence of major vertical gradients within the leaf is often overlooked in studies of photosynthesis. These gradients, which involve light heterogeneity, cell composition, and CO₂ concentration across the mesophyll, can generate differences in the maximum potential PSII efficiency (F _V/F _M or F _V/F _P) of the different cell layers. Evidence is presented for a step gradient of F _V/F _P ratios across the mesophyll, from the adaxial (palisade parenchyma, optimal efficiencies) to the abaxial (spongy parenchyma, sub-optimal efficiencies) side of Quercus coccifera leaves. For this purpose, light sources with different wavelengths that penetrate more or less deep within the leaf were employed, and measurements from the adaxial and abaxial sides were performed. To our knowledge, this is the first report where a low photosynthetic performance in the abaxial side of leaves is accompanied by impaired F _V/F _P ratios. This low photosynthetic efficiency of the abaxial side could be related to the occurrence of bundle sheath extensions, which facilitates the penetration of high light intensities deep within the mesophyll. Also, leaf morphology (twisted in shape) and orientation (with a marked angle from the horizontal plane) imply direct sunlight illumination of the abaxial side. The existence of cell layers within leaves with different photosynthetic efficiencies makes appropriate the evaluation of how light penetrates within the mesophyll when using Chl fluorescence or gas exchange techniques that use different wavelengths for excitation and/or for driving photosynthesis.     

Separating the contribution of the upper and lower mesophyll to photosynthesis in Zea mays L. leaves

The appearance of transverse sections of maize leaves indicates the existence of two airspace systems serving the mesophyll, one connected to the stomata of the upper epidermis and the other to the stomata of the lower surface, with few or no connections between the two. This study tests the hypothesis that the air-space systems of the upper and lower mesophyll are separated by a defined barrier of measurable conductance. A mathematical procedure, based on this hypothesis, is developed for the quantitative separation of the contributions made by the upper and lower halves of the mesophyll to carbon assimilation using gasexchange data. Serial paradermal sections and three-dimensional scanning-electron-microscope images confirmed the hypothesis that there were few connections between the two air-systems. Simultaneous measurements of nitrous-oxide diffusion across the leaf and of transpiration from the two surfaces showed that the internal conductance was about 15% of the maximum observed stomatal conductance. This demonstrates that the poor air-space connections, indicated by microscopy, represent a substantial barrier to gas diffusion. By measuring the CO₂ and water-vapour fluxes from each surface independently, the intercellular CO₂ concentration (c _i) of each internal air-space system was determined and the flux between them calculated. This allowed correction of the apparent CO₂ uptake at each surface to derive the true CO₂ uptake by the mesophyll cells of the upper and lower halves of the leaf. This approach was used to analyse the contribution of the upper and lower mesophyll to CO₂ uptake by the leaf as a whole in response to varying light levels incident on the upper leaf surface. This showed that the upper mesophyll was light-saturated by a photon flux of approx. 1000 mol·m^-2·s^-1 (i.e. about one-half of full sunlight). The lower mesophyll was not fully saturated by photon fluxes of nearly double full sunlight. At low photon fluxes the c _i of the upper mesophyll was significantly less than that of the lower mesophyll, generating a significant upward flux of CO₂. At light levels equivalent to full sunlight, and above, c _i did not differ significantly between the two air space systems. The physiological importance of the separation of the air-space systems of the upper and lower mesophyll to gas exchange is discussed.Abbreviations and symbols A net leaf CO₂ uptake rate - A _upper ^app. and A _lower ^app. net rates of CO₂ uptake across the upper and lower surfaces - A _upper and A _lower derived net rates of CO₂ uptake by the upper and lower mesophyll - A _upward net flux of CO₂ from the lower to upper mesophyll - c _a, c _{a, upper} and c _{a, lower} the CO₂ concentrations in the air around the leaf above the upper surface and below the lower surface - c _N2O the concentration of N₂O in the air around the leaf - c _i, c _{i, upper} and c _{i, lower} the mesophyll intercellular CO₂ concentration of the whole leaf, the upper mesophyll and the lower mesophyll - g _i leaf internal conductance to CO₂ - g _s, g _{s, lower} and g _{s, upper} the stomatal conductance of the whole leaf, the lower surface and the upper surface - g the total conductance across the leaf - Q the photosynthetically active photon flux density     

Untangling metabolic and spatial interactions of stress tolerance in plants. 1. Patterns of carbon metabolism within leaves

The localization of the key photoreductive and oxidative processes and some stress-protective reactions within leaves of mesophytic C₃ plants were investigated. The role of light in determining the profile of Rubisco, glutamate oxaloacetate transaminase, catalase, fumarase, and cytochrome-c-oxidase across spinach leaves was examined by exposing leaves to illumination on either the adaxial or abaxial leaf surfaces. Oxygen evolution in fresh paradermal leaf sections and CO₂ gas exchange in whole leaves under adaxial or abaxial illumination was also examined. The results showed that the palisade mesophyll is responsible for the midday depression of photosynthesis in spinach leaves. The photosynthetic apparatus was more sensitive to the light environment than the respiratory apparatus. Additionally, examination of the paradermal leaf sections by optical microscopy allowed us to describe two new types of parenchyma in spinach—pirum mesophyll and pillow spongy mesophyll. A hypothesis that oxaloacetate may protect the upper leaf tissue from the destructive influence of active oxygen is presented. The application of mathematical modeling shows that the pattern of enzymatic distribution across leaves abides by the principle of maximal ecological utility. Light regulation of carbon metabolism across leaves is discussed.     

Ontogenetic changes in stomatal size and conductance of sunflowers

The ontogenetic changes in stomatal size, frequency and conductance (g_s) on abaxial and adaxial leaf surfaces of sunflower plants (Helianthus annuus L. Russian Mammoth) were examined under controlled environmental conditions. The stomatal frequency on the adaxial and abaxial leaf surfaces decreased with leaf ontogeny and insertion level. The ratio of adaxial to abaxial stomatal frequency did not change with leaf ontogeny and insertion level, and 42–44% of total stomata was apportioned to the adaxial surface. Ontogenetic changes in stomatal pore length were detected and increased with ontogenesis. The stomatal length of both leaf surfaces had linear relationships with leaf area. Ontogenetic changes in g_s were similar between the two surfaces. However the adaxial g_s was lower than abaxial g_s in leaves of higher insertion levels. Conductance had a linear relationship with width x frequency but not with pore area.     

Leaf traits variation during leaf expansion in <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The morphological, anatomical and physiological variations of leaf traits were analysed during Quercus ilex L. leaf expansion. The leaf water content (LWC), leaf area relative growth rate (RGR_l) and leaf dry mass relative growth rate (RGR_m) were the highest (76±2 %, 0.413 cm² cm⁻² d⁻¹, 0.709 mg mg⁻¹ d⁻¹, respectively) at the beginning of the leaf expansion process (7 days after bud break). Leaf expansion lasted 84±2 days when air temperature ranged from 13.3±0.8 to 27.6±0.9 °C. The net photosynthetic rate (P _N), stomatal conductance (g _s), and chlorophyll content per fresh mass (Chl) increased during leaf expansion, having the highest values [12.62±1.64 μmol (CO₂) m⁻² s⁻¹, 0.090 mol (H₂O) m⁻² s⁻¹, and 1.03±0.08 mg g⁻¹, respectively] 56 days after bud break. Chl was directly correlated with leaf dry mass (DM) and P _N. The thickness of palisade parenchyma contributed to the total leaf thickness (263.1±1.5 μm) by 47 %, spongy layer thickness 38 %, adaxial epidermis and cuticle thickness 9 %, and abaxial epidermis and cuticle thickness 6 %. Variation in leaf size during leaf expansion might be attributed to a combination of cells density and length, and it is confirmed by the significant (p<0.001) correlations among these traits. Q. ilex leaves reached 90 % of their definitive structure before the most severe drought period (beginning of June — end of August). The high leaf mass area (LMA, 15.1±0.6 mg cm⁻²) at full leaf expansion was indicative of compact leaves (2028±100 cells mm⁻²). Air temperature increasing might shorten the favourable period for leaf expansion, thus changing the final amount of biomass per unit leaf area of Q. ilex.     

Stomatal function,density and pattern,and CO2 assimilation in Arabidopsis thaliana tmm1 and sdd1‐1 mutants

• Stomata modulate the exchange of water and CO₂ between plant and atmosphere. Although stomatal density is known to affect CO₂ diffusion into the leaf and thus photosynthetic rate, the effect of stomatal density and patterning on CO₂ assimilation is not fully understood.
• We used wild types Col‐0 and C24 and stomatal mutants sdd1‐1 and tmm1 of Arabidopsis thaliana, differing in stomatal density and pattern, to study the effects of these variations on both stomatal and mesophyll conductance and CO₂ assimilation rate. Anatomical parameters of stomata, leaf temperature and carbon isotope discrimination were also assessed.
• Our results indicate that increased stomatal density enhanced stomatal conductance in sdd1‐1 plants, with no effect on photosynthesis, due to both unchanged photosynthetic capacity and decreased mesophyll conductance. Clustering (abnormal patterning formed by clusters of two or more stomata) and a highly unequal distribution of stomata between the adaxial and abaxial leaf sides in tmm1 mutants also had no effect on photosynthesis.
• Except at very high stomatal densities, stomatal conductance and water loss were proportional to stomatal density. Stomatal formation in clusters reduced stomatal dynamics and their operational range as well as the efficiency of CO₂ transport.

     

Photosynthetic CO2 uptake byZea mays leaves as influenced by unilateral irradiation of adaxial and abaxial leaf surfaces

A study was made on the effect of increasing photon fluence rate (I) at a unilateral irradiation of adaxial (normal leaf position) and abaxial (inverse leaf position) blade surface of maize leaves of various insertion levels on net photosynthetic CO₂ uptake (P _n ) by the leaves, as well as the contribution of individual surfaces toP _n of the leaves, and the significance of, or relationship between the stomatal (g _s ) and intracellular (gm) conductances at the CO₂ transport.P _n of leaves of various age according to their insertion level was unaffected by the direction of incident irradiation. Upon irradiation of the leaves in normal and inverse position the contribution of the adaxial and abaxial surfaces toP _n ,g _s and g_m was different. On irradiating the leaves in normal position, the contribution of the irradiated adaxial surface to the characteristics mentioned made on the average 55% of total values, the contribution of the abaxial surface irradiated in inverse position made on the average 70% inP _n andg _m , and 80% ing _s . At lowerI’s g _m was higher thang _s both in irradiated and non-irradiated surfaces. The ratio ofg _s to g_m gradually got square with increasingI. In the irradiated adaxial surface the equilibrium (g _s /g _m = 1.0) took place at the highestI’s, in the irradiated abaxial surface between 500 to 1000 μmol m⁻² s⁻¹. The significance of the ratiog _m in the CO₂ transport through the individual surfaces is discussed.     

CO2 transfer conductance, leaf structure and carbon isotope composition of Polygonum cuspidatum leaves from low and high altitudes

Anatomy and some physiological characteristics of the leaves in Polygonum cuspidatum Sieb. et Zucc., a dioecious clonal herb, were compared between two populations, one from a lowland in Shizuoka City (10 m above sea level), and another from a highland on Mt. Fuji (2500 m above sea level). Leaf mass per area (LMA) of the highland plants was about twice that of the lowland plants. The greater leaf thickness, thicker mesophyll cell walls and higher mesophyll cell density in the highland leaves contributed to the larger LMA. Although mesophyll area exposed to intercellular airspaces was greater in the highland leaves than in the lowland leaves by 30%, the surface area of chloroplasts facing intercellular airspaces was similar between these leaves. CO₂ transfer conductance inside the leaf (g_i) of the highland leaves (0·75 μmol m^?2 s^?1 Pa^?1) is the lowest recorded for herbaceous plants and was only 40% of that in the lowland leaves. On the other hand, the difference in stomatal conductance was small. δ¹³C values in the leaf dry matter were greater in the highland leaves by 4‰. These data and the estimation of CO₂ partial pressures in the intercellular air spaces and in the chloroplast suggested that the greater dry matter δ¹³C in the highland leaves, indicative of lower long‐term ratio of the chloroplast stroma to the ambient CO₂ partial pressures, would be mainly attributed to their lower g_i.     

δ13C of CO2 respired in the dark in relation to δ13C of leaf metabolites: comparison between Nicotiana sylvestris and Helianthus annuus under drought

The variations of δ¹³C in leaf metabolites (lipids, organic acids, starch and soluble sugars), leaf organic matter and CO₂ respired in the dark from leaves of Nicotiana sylvestris and Helianthus annuus were investigated during a progressive drought. Under well‐watered conditions, CO₂ respired in the dark was ¹³C‐enriched compared to sucrose by about 4‰ in N. sylvestris and by about 3‰ and 6‰ in two different sets of experiments in H. annuus plants. In a previous work on cotyledonary leaves of Phaseolus vulgaris, we observed a constant ¹³C‐enrichment by about 6‰ in respired CO₂ compared to sucrose, suggesting a constant fractionation during dark respiration, whatever the leaf age and relative water content. In contrast, the ¹³C‐enrichment in respired CO₂ increased in dehydrated N. sylvestris and decreased in dehydrated H. annuus in comparison with control plants. We conclude that (i) carbon isotope fractionation during dark respiration is a widespread phenomenon occurring in C₃ plants, but that (ii) this fractionation is not constant and varies among species and (iii) it also varies with environmental conditions (water deficit in the present work) but differently among species. We also conclude that (iv) a discrimination during dark respiration processes occurred, releasing CO₂ enriched in ¹³C compared to several major leaf reserves (carbohydrates, lipids and organic acids) and whole leaf organic matter.     

Influence of growth regulators on direct embryo formation from leaf explants of Phalaenopsis orchids

Leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were used to test the effects of auxins (2,4-D, IAA, IBA, NAA), cytokinins (2iP, BA, kinetin, TDZ, zeatin), GA₃, ancymidol, polyamines (putrescine, spermine, spermidine), ACC, AgNO₃ and CoCl₂ on the amount of direct embryo formation on different leaf locations (the cut end, the adaxial side, the abaxial side and the leaf tip). The results showed that there was a genotypic effect on direct embryo formation induced by cytokinins that 13.32 μM BA and 4.92 μM 2iP was the most effective in P. amabilis and P. Nebula, respectively. Besides, explant position highly affected embryogenic competence of leaf cells in both species that the cut end showed highest embryogenic response, the adaxial side was the second, and then the abaxial side and the leaf tip. Altogether, cytokinins tested were all effective in both species, and ACC at 20 μM had 35% of embryogenic response in P. amabilis. However, auxins, GA₃, ancymidol and polyamines were inhibitory in both species.     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.     

Studies on octylphenoxy surfactants: IX. Effect of oxyethylene chain length on GA3 absorption by sour cherry leaves

The effect of a homologous series of octylphenoxy surfactants, -[4-(1,1,3,3-tetramethylbutyl)phenyl]--hydroxypoly-(oxy-1,2-ethanediyl), condensed with 5, 7–8, 9–10, 16, and 30 oxyethylene (EO) units on enhancement of gibberellic acid (GA₃) absorption by leaves ofPrunus cerasus cv. Montmorency was studied. Increasing EO chain length (5–30 EO) increased surface tension (27.5–35.3 mN m^–1) and contact angles on adaxial (21–36°) and abaxial (28–49°) leaf surfaces. With increasing EO content, the form of GA₃ deposits from droplets on the leaf surface changed from an annulus shape (5 and 7–8 EO) to globular forms covering increasingly smaller interface areas (9–10 to 30 EO). The surfactants increased GA₃ uptake, the magnitude decreased with an increase in oxyethylene chain length. Similar trends were found for both the adaxial and abaxial surfaces. Penetration through the abaxial surface was linearly related to the logarithm of the oxyethylene content of the surfactant molecule (r ²=0.934^**) and to the hydrophilic: lipophilic balance (r ²=0.926^**). Absorption by the abaxial surface was approximately one order of magnitude greater than by the adaxial surface.     

High-light effects on CO2 fixation gradients across leaves

Chlorophyll fluorescence and internal patterns of ¹⁴CO₂ fixation were measured in sun and shade leaves of spinach after treatment with various light intensities. When sun leaves were irradiated with 2000μmol m^?2 s^?1 for 2h, F_V/F_M decreased by about 15%, but ¹⁴CO₂ fixation was unaffected, whereas shade leaves exhibited a 21% decrease in F_v/F_M and a 25% decrease in ¹⁴CO₂ fixation. Irradiation of sun and shade leaves with 4000μmol m^?1 for 4 h decreased F_V/F_M by 30% in sun leaves and 40% in shade leaves, while total ¹⁴CO₂ fixation decreased by 41% in sun leaves and 55% in shade leaves. After light treatment, gradients of CO₂ fixation across leaves were determined by measuring ¹⁴CO₂ fixed in paradermal leaf sections after a 10s pulse of ¹⁴CO₂. Gradients of ¹⁴CO₂ fixation in control sun and shade leaves were identified when expressed on a relative basis and normalized for leaf depth. Treatment of leaves with 2000 μmol PAR m^?2 s^?1 for 2h did not after patterns of carbon fixation across sun leaves, but slightly altered the pattern in shade leaves. In contrast, treatment of sun and shade leaves with 4000μmol m^?2 s^?1 for 4h decreased carbon fixation more in the palisade mesophyll cells than in the spongy mesophyll cells of sun and shade leaves, and fixation in medial tissue of shade leaves was dramatically decreased compared to the adaxial and abaxial tissue. The interaction between leaf anatomy and biochemical parameters involved in tolerance to photoinhibition in spinach is discussed.