Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue

The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var `Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells.     

Responses of Photosynthetic Electron Transport in Stomatal Guard Cells and Mesophyll Cells in Intact Leaves to Light, CO2, and Humidity

High-resolution images of the chlorophyll fluorescence parameter Fq'/Fm' from attached leaves of commelina (Commelina communis) and tradescantia (Tradescantia albiflora) were used to compare the responses of photosynthetic electron transport in stomatal guard cell chloroplasts and underlying mesophyll cells to key environmental variables. Fq'/Fm' estimates the quantum efficiency of photosystem II photochemistry and provides a relative measure of the quantum efficiency of non-cyclic photosynthetic electron transport. Over a range of light intensities, values of Fq'/Fm' were 20% to 30% lower in guard cell chloroplasts than in mesophyll cells, and there was a close linear relationship between the values for the two cell types. The responses of Fq'/Fm' of guard and mesophyll cells to changes of CO2 and O2 concentration were very similar. There were similar reductions of Fq'/Fm' of guard and mesophyll cells over a wide range of CO2 concentrations when the ambient oxygen concentration was decreased from 21% to 2%, suggesting that both cell types have similar proportions of photosynthetic electron transport used by Rubisco activity. When stomata closed after a pulse of dry air, Fq'/Fm' of both guard cell and mesophyll showed the same response; with a marked decline when ambient CO2 was low, but no change when ambient CO2 was high. This indicates that photosynthetic electron transport in guard cell chloroplasts responds to internal, not ambient, CO2 concentration.     

The functional significance of palisade tissue: penetration of directional versus diffuse light

Light gradients were measured in leaves that had different types of anatomical development of the mesophyll but similar pigment content. Leaves of the legume, Thermopsis montana, had columnar palisade and spongy mesophyll whereas leaves of the monocot, Smilacina stellata, had spongy mesophyll only. Light gradients were measured at 550 nm in both types of leaves when they were irradiated with collimated or diffuse light. When irradiated with collimated light, light gradients were steeper in leaves with spongy mesophyll in comparison to those that had palisade tissue. On the other hand, light gradients were similar between both leaf types when they were irradiated with diffuse light. Thus, columnar palisade cells facilitated the penetration of collimated light over diffuse light. These results suggest that palisade tissue may help distribute light more uniformly to chloroplasts within the leaf. Moreover, the functional significance of palisade tissue may be related to the amount of collimated light within the natural environment.     

Profiles of photosynthetic oxygen-evolution within leaves of Spinacia oleracea

Oxygen evolution was measured from mesophyll tissues in spinach leaves using a photoacoustic technique. The photosynthetic capacity of individual cell layers was measured by directing microscopic beams of light, 40 μm wide, to cells exposed within a leaf cross section. The resulting profile for oxygen-evolution potential was relatively flat, indicating a uniform capacity for photosynthesis in leaf mesophyll tissues. Two experimental approaches were used to estimate the photosynthetic performance of individual mesophyll cell layers when white light was applied to the adaxial leaf surface. These experiments indicated that oxygen was produced relatively uniformly across the mesophyll and that oxygen evolution increased with irradiance of the white light applied to the leaf surface. The measured profiles for oxygen evolution and capacity are flatter than previous measurements of profiles of fixed carbon and estimates of profiles for absorbed light within spinach leaves.     

Identification and localization of vegetative STORAGE PROTEINS IN LEGUME LEAVES

Leaves from 12 legume species representing two subtribes were examined by various techniques for the presence of vegetative storage proteins (VSPs) similar to the 27, 29, and 94 kD VSPs of soybean. Polyacrylamide gel electrophoresis (PAGE) of leaf protein followed by western immunoblotting using antibody that recognizes soybean VSP94, a lipoxygenase, demonstrated that this protein is present in six of the nine species tested. Blotting with antibody to soybean VSP27/29, which are glycoproteins, gave labelling in seven species and glycoprotein affino-blots showed that glycosylated proteins ranging around 27 to 29 kD were present in all nine species examined. Immunocytochemical localization studies of eight species demonstrated that proteins antigenically similar to VSP94 and VSP27/29 are specifically accumulated in the vacuole of paraveinal mesophyll (PVM) cells. They were not detectable at significant levels in other mesophyll cells using this technique. Comparisons of protein compositions of isolated PVM and mesophyll protoplasts from seven species further confirmed the specialized nature of the PVM. VSP94 and proteins ranging from 25 to 35 kD molecular mass were the major proteins of PVM of all but one species while Rubisco was quite low in amount compared to mesophyll protoplasts. The results show that VSP synthesis and accumulation is a general feature of legume leaves containing a PVM layer and indicate that the PVM plays a specialized role in nitrogen metabolism and partitioning in these species.     

Mesophyll protoplasts of fenugreek (Trigonella foenumgraecum): Isolation,culture and shoot regeneration

Plant Cell Reports - High yields of mesophyll protoplasts were obtained after treatment of leaves of Trigonella foenumgraecum, a forage legume, with purified cellulase. Under appropriate conditions...     

On the Resistance to Transpiration of the Sites of Evaporation within the Leaf

The rates of transpiration from the upper and lower surfaces of leaves of Gossypium hirsutum, Xanthium strumarium, and Zea mays were compared with the rates at which helium diffused across those leaves. There was no evidence for effects of CO₂ concentration or rate of evaporation on the resistance to water loss from the evaporating surface (“resistance of the mesophyll wall to transpiration”) and no evidence for any significant wall resistance in turgid tissues. The possible existence of a wall resistance was also tested in leaves of Commelina communis and Tulipa gesneriana whose epidermis could be easily peeled. Only when an epidermis was removed from a leaf, evaporation from the mesophyll tissue declined. We conclude that under conditions relevant to studies of stomatal behavior, the water vapor pressure at the sites of evaporation is equal to the saturation vapor pressure.     

Evidence for involvement of photosynthetic processes in the stomatal response to CO2

Stomatal conductance (gs) typically declines in response to increasing intercellular CO2 concentration (ci). However, the mechanisms underlying this response are not fully understood. Recent work suggests that stomatal responses to ci and red light (RL) are linked to photosynthetic electron transport. We investigated the role of photosynthetic electron transport in the stomatal response to ci in intact leaves of cocklebur (Xanthium strumarium) plants by examining the responses of gs and net CO2 assimilation rate to ci in light and darkness, in the presence and absence of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and at 2% and 21% ambient oxygen. Our results indicate that (1) gs and assimilation rate decline concurrently and with similar spatial patterns in response to DCMU; (2) the response of gs to ci changes slope in concert with the transition from Rubisco- to electron transport-limited photosynthesis at various irradiances and oxygen concentrations; (3) the response of gs to ci is similar in darkness and in DCMU-treated leaves, whereas the response in light in non-DCMU-treated leaves is much larger and has a different shape; (4) the response of gs to ci is insensitive to oxygen in DCMU-treated leaves or in darkness; and (5) stomata respond normally to RL when ci is held constant, indicating the RL response does not require a reduction in ci by mesophyll photosynthesis. Together, these results suggest that part of the stomatal response to ci involves the balance between photosynthetic electron transport and carbon reduction either in the mesophyll or in guard cell chloroplasts.     

Chloroplast-located flavonoids can scavenge singlet oxygen

* The hypothesis was tested that flavonoids may scavenge singlet oxygen ((1)O(2)) in mesophyll cells of Phillyrea latifolia exposed to excess-light stress. * In cross-sections taken from leaves developed at 10% (shade) or 100% (sun) solar irradiance, we evaluated the excess photosynthetically active radiation (PAR)-induced accumulation of (1)O(2) in mesophyll cells by imaging the fluorescence quenching of the specific (1)O(2) probe N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy). The intracellular location of flavonoids was also analyzed using three-dimensional deconvolution microscopy. * Photo-induced quenching of DanePy fluorescence was markedly greater in the mesophyll of shade leaves than in that of sun leaves, the former showing a negligible accumulation of mesophyll flavonoids. The photo-induced generation of (1)O(2) was inversely related to the content of flavonoids in the mesophyll cells of sun leaves. Flavonoids were located in the chloroplasts, and were likely associated with the chloroplast envelope. * Here we provide relevant evidence for the potential scavenger activity of chloroplast-located flavonoids against (1)O(2) and new insights into the photo-protective role of flavonoids in higher plants.     

Determination of mesophyll diffusion resistance in Chamaerion angustifolium by the method of three-dimensional reconstruction of the leaf cell packing

A detailed quantitative analysis of the three-dimensional organization of the mesophyll was performed, and mesophyll diffusion resistance to CO₂ in the leaves of Chamaerion angustifolium formed under different irradiance was calculated using an original method of stereometric cellular packing. For each type of leaves (sun and shade), we determined structural components of gas exchange: the volume of mesophyll per unit leaf area (V _mes), the volume of the intercellular space in the mesophyll (V _is), the area of the total mesophyll surface (S), the area of the free mesophyll surface facing the intercellular spaces (S _mes), and the ratios of the total and the free mesophyll surfaces to its volume (S/V and S _mes/V). As compared with sun leaves, in the shade leaves of Ch. angustifolium, S and V _mes decreased twofold, tissue density was reduced twofold, and the share of the intercellular space in the mesophyll rose from 49 to 72%. In shade, the diffusion resistance of the mesophyll increased by 1.8 times because of changes in the leaf structure. At the same time, the ratio S _mes/V was found to increase by 1.4 times, which facilitated the diffusion of CO₂. In the shade leaves of Ch. angustifolium, the diffusion resistance of the intercellular air spaces was reduced twofold as a result of an increase in their share in the leaf mesophyll and simplification of their geometry. Thus, the method of three-dimensional reconstruction of sun and shade leaves of Ch. angustifolium showed a comprehensive rearrangement of the mesophyll spatial organization in shade and revealed the structural mechanisms of changes in the resistance to CO₂ diffusion within the leaf.     

Effect of Several Transpiration Suppressants on Carbon Dioxide and Water Vapor Exchange of Citrus and Grapevine Leaves

Several transpiration suppressants, (phenyl mercuric acetate, Tag 16 (polyethylen emulsion) R₁₄-poly (ethylene adipate) 4–4 Diphenylmethan diisocyanate), which are known to either close stomata or form thin films on leaves, were sprayed on citrus and grapevine seedlings. Water vapor and carbon dioxide exchange of single leaves were measured by means of infra red gas analyzer and L1C1 hygrosensors. The effects of the chemicals were evaluated by analyzing net photosynthesis, transpiration, mesophyll resistance to CO₂ compensation concentration and respiration. All the chemicals tested increased either mesophyll resistance or CO₂ compensation point to various degrees. It is concluded that none of the chemicals tested acted solely on epidermal resistance. All chemicals reduced photosynthesis, but the ratio of photosynthesis to transpiration increased in most cases in grapevine but not in citrus. Different chemicals acted differently on citrus and grapevine. The method of analysis used enabled us to evaluate separately the relative effect of a chemical on mesophyll resistance and respiration.     

Study of Diurnal Changes in Photosynthetic Rate and Quantum Efficiency of Grapevine Leaves and Their Utilization in Canopy Management

Photosynthetic rate and quatum efficiency of grapevine (Vitis vinifera L. cv. Sauvignon blanc) leaves were measured under the field with ample soil water supply, and in phytotron with ample supply of water and mineral nutrients, constant air humidity and CO2 concentration, and optimum air temperature, respectively. Under field conditions CO2 assimilation quantum efficiency of leaves reached its maximum in the morning, which was followed by continuous decrease and midday depression. The leaves intercepting more light energy in the morning showed a higher quantum efficiency. Those leaves subjected continuously to strong irradiance exhibited a more obvious and longer midday depression. Reduction of leaf light interception around midday could reduce midday depression. Shaded leaves had a higher quantum efficiency than leaves under direct sunlight. The diurnal changes in photosynthetic rate and quantum efficiency of leaves were shown to be closely related to the variations in mesophyll resistance to CO2. In phytotron experiments the photosynthetic quantum efficiency of leaves was reduced after a certain period of illumination not only at 1200 μmol · m-2 · s-1 PFD, higher than the saturating light of vine leaves (≈1000 μmol · m-2 · s-1), which was caused by "photoinhibition”, but also at 800 and 200μmol · m-2 · s-1, which was similar to "photoinhibition”. But photosynthetic quantum efficiency of leaves exposed continuously to a very weak PFD (100 μmol · m -2 · s-1) remained contant. The diurnal changes in mesophyll resistance to CO2 of vine leaves could be partly related to photoinhibition. It is considered that, under field conditions without soil water limitation, midday depression of vine leaf photosynthesis could be a result of an increase of the mesophyll resistance induced by multiple effects of strong light, high temperature and low humidity. A higher light interception by canopy plane in the morning may be advantageous to exploit higher photosynthetic potentiality of leaves, but a lower light interception in the middle of day may reduce midday depression. The north-south orientation plane can provide optimum light regime and improve photosynthetic environment in vineyards.     

Changes in the Resistance of the Mesophyll Tissue 1

Porometer measurements of the resistance offered to viscousflow of air by mesophyll tissue rings cut from leaves were carriedout simultaneously with determinations of changes in tissuewater content, Decreases in water content were accompanied byincreases in mesophyll resistance and these changes are discussedin relation to porometer measurements of total resistance offeredby leaves to viscous flow of air.     

Effects of Powdery Mildew Infection on the Efficiency of CO(2) Fixation and Light Utilization by Sugar Beet Leaves

Sugar beet leaves (Beta vulgaris L.) infected with powdery mildew (Erysiphe polygoni D.C.) show declining rates of net photosynthesis as the disease develops; relative to healthy controls, reductions of 35, 70, and 75% were observed at 9, 16, and 22 days after inoculation, respectively. A leaf gas exchange procedure in which an air stream flowed through the leaf showed that mesophyll conductance declined in parallel with photosynthesis in mildew-infected leaves. Viscous flow conductance of diseased leaves also declined over the same period suggesting that stomatal aperture was reduced. From the magnitude and time course of disease effects on stomatal aperture and mesophyll conductance, it appears that the effects at the mesophyll level were primarily responsible for mediating the decline in net photosynthesis. Changes in mesophyll conductance were closely correlated with reduced activity of ribulose-1,5-bisphosphate carboxylase on a leaf area basis. This decrease could be attributed to a reduction in the concentration of the enzyme, a reduction which was greater than the reduction in total soluble protein. The quantum efficiency of light use was also decreased by the disease. Mildew-infected leaves had quantum yields that were reduced, relative to healthy leaves, by 17 and 22% at 14 and 18 days after inoculation, respectively.     

Evidence for Hygrometric Pressurization in the Internal Gas Space of Spartina alterniflora

Higher pressure, up to several hundred pascal relative to ambient, is generated by hygrometric pressurization within the central hollow space of the stem in Spartina alterniflora. Dilution of oxygen and nitrogen by water vapor within the plant's internal gas space results in an influx of nitrogen and oxygen from the air and a net increase in the internal gas pressure at steady state. The nature of the pressure gradient suggests that small pores exist in the plant tissues. Moreover, the compact arrangement of leaf mesophyll cells creates a high resistance for the mass flow of gases and contributes to the higher pressure within leaves. After experimentally venting the internal pressure, outside air diffused through the basal area of the adaxial side of the leaves into the internal space and elevated pressure was restored.     

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.     

Estimation of Photorespiration of Green Plants and of their Mesophyll Resistance to CO2 Uptake

An estimate of photorespiration is obtained from the relationshipbetween the net exchange of CO₂ of the leaf and the internalCO₂ concentration, i.e. within the mesophyll intercellular spaces.The latter is obtained by calculation, taking into account thecombined epidermal and boundary-layer resistances between thebulk atmosphere and the mesophyll intercellular spaces. Thelinear part of this relationship (at low CO₂ concentrations)is extrapolated to zero internal concentration, at which noneof the intercellular photorespired CO₂ is available for reassimilation.The calculated output of CO₂ under such conditions providesan estimate of photorespiration, but, by failing to take intoaccount intracellular reassimilation of photorespired CO₂ underestimatesactual photorespiration. As the slope of this linear relationshiprepresents the mesophyll (intracellular) resistance to CO₂ uptake,this procedure was used to recalculate published data on effectsof light intensity and of oxygen concentration on net photosynthesis.The analysis showed that increased oxygen concentration anddecreased light intensity reduced photosynthesis largely byincreasing mesophyll resistance to CO₂ uptake. It is suggestedthat the CO₂ compensation point () is a function of both photorespiration(L) and mesophyll resistance (r_m): = L. r_m.     

Induced Systemic Resistance in Tomato Plants against Phytophthora infestans

Infection of lower leaves of tomato plants with Phytophthora infestans followed by a period unfavourable to disease development increased the general resistance of the plants against the pathogen. Induced resistance to a second infection of upper leaves was expressed in the development of necrotic lesions that were sharply defined and reduced in size. Sporulation of the pathogen was suppressed. Lesions on unprotected plants expanded with a sporulating zone passing gradually to the healthy tissue under the same conditions. Induced resistance delayed and reduced penetration of the pathogen into the epidermis and subsequent colonisation of the mesophyll by formation of papillae in epidermal cells and hypersensitive-like reactions of penetrated, mesophyll cells.     

Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source

The hypothesis that anthocyanins in red leaves may be potential in vivo antioxidants whose efficiency is linked to their proximity with the oxy-radical source was tested. Advantage was taken of intra-individual and intra-species variations in the anthocyanic trait and green and red leaves on the same individuals or leaves of green and red phenotypes were compared for the extent of PSII damage by reactive oxygen species generated by methyl viologen treatment in the light. Two species possessing anthocyanins in the mesophyll (Cistus creticus and Photinia x fraseri) and two in the epidermis (Rosa sp. and Ricinus communis) were used, while red actinic light (which is not absorbed by anthocyanins) allowed discrimination between an indirect sunscreen and a direct antioxidant function. Red leaves whose anthocyanins were located in the mesophyll were more resistant to methyl viologen treatment than their green counterparts. In one of these species (Cistus creticus), where anthocyanins are induced in some individuals within the natural population after bright cool days in winter, both green and future-red morphs displayed the same sensitivity to methyl viologen before anthocyanin induction. Immediately after reddening, however, resistance to methyl viologen was considerably increased in the red morphs. By contrast, red leaves whose anthocyanins were restricted to epidermal cells were more sensitive to the herbicide. Total leaf phenolic levels in green/red pairs were similar. The results indicate that vacuolar anthocyanins may be an effective in vivo target for oxy-radicals, provided that the oxy-radical source and the anthocyanic detoxifying sink are in close vicinity.     

Interactions among leaf toughness, chemistry, and harvesting by attine ants

Abstract. 1. Young and mature leaves of a tropical legume, Inga edulis var. minutula Schery, are strikingly different in secondary chemistry, especially condensed tannins, and leaf toughness.
2. Bioassays with the two different leaf types indicate that leaf cutter ants, Atta cephalotes (L.), always find mature leaves relatively more acceptable than young leaves when selection was based on chemical cues.
3. Since extracts of young leaves show greziter inhibition of fungal pectinases we suggest that leaf-cutter ants are capable of distinguishing which leaf types are most suitable for the growth of their symbiotic fungus.
4. However, mature leaves are 3 times tougher than young leaves and this prevents leaf-cutter ants from harvesting the more suitable mature leaves.
5. Consequently, bioassays which require cutting before leaf removal indicate that some colonies actually harvest more from the less suitable young leaves.
6. We suggest that the quality of a colony's habitat may indicate whether a colony will harvest more of the less suitable young leaves. Colonies which are harvesting from highly suitable host plants avoid the tropical legume I.edulis while those in poorer habitats accept I.edulis but, because of leaf toughness, mostly harvest the less suitable young leaves.