Partitioning of Photosynthetically Fixed 14CO2 Into Oil and Curcumin Accumulation in Curcuma Longa Grown Under Iron Deficiency

Changes in leaf growth, photosynthetic efficiency, and incorporation pattern of photosynthetically fixed ¹⁴CO₂ in leaves 1 and 2 from plant apex, in roots, and rhizome induced in Curcuma by growing in a solution culture at Fe concentration of 0 and 5.6 g m^–3 were studied. ¹⁴C was incorporated into primary metabolites (sugars, amino acids, and organic acids) and secondary metabolites (essential oil and curcumin). Fe deficiency resulted in a decrease in leaf area, its fresh and dry mass, chlorophyll (Chl) content, and CO₂ exchange rate at all leaf positions. The rate of ¹⁴CO₂ fixation declined with leaf position, maximum being in the youngest leaf. Fe deficiency resulted in higher accumulation of sugars, amino acids, and organic acids in leaves at both positions. This is due to poor translocation of metabolites. Roots and rhizomes of Fe-deficient plants had lower concentrations of total photosynthate, sugars, and amino acids whereas organic acid concentration was higher in rhizomes. ¹⁴CO₂ incorporation in essential oil was lower in the youngest leaf, as well as incorporation in curcumin content in rhizome. Fe deficiency influenced leaf area, its fresh and dry masses, CO₂ exchange rate, and oil and curcumin accumulation by affecting translocation of assimilated photosynthates.     

Refixation of xylem sap CO2 in Populus deltoides

Vascular plants have respiring tissues which are perfused by the transpiration stream, allowing solubilization of respiratory CO₂ in the xylem sap. The transpiration stream could provide a conduit for the internal delivery of respiratory CO₂ to leaves. Trees have large amounts of respiring tissues in the root systems and stems, and may have elevated levels of CO₂ in the xylem sap which could be delivered to and refixed by the leaves. Xylem sap from the shoots of three Populus deltoides trees had mean dissolved inorganic carbon concentrations (CO₂+H₂CO₃+HCO^?₃) ranging from 0. 5 to 0. 9 mM. When excised leaves were allowed to transpire 1 mM[¹⁴C]NaHCO₃, 99. 6% of the label was fixed in the light. Seventy-seven percent of the label was fixed in major veins and the remainder was fixed in the minor veins. Autoradiography confirmed that label was confined to the vasculature. In the dark, approximately 80% of the transpired label escaped the leaf, the remainder was fixed in the major veins, slightly elevating dark respiration measurements. This indicates that the vascular tissue in P. deltoides leaves is supplied with a carbon source distinct from the atmospheric source fixed by interveinal lamina. However, the contribution of CO₂ delivered to the leaves in the transpiration stream and fixed in the veins was only 0. 5% of atmospheric CO₂ uptake. In the light 90% of the label was found in sugar, starch and protein, a pattern similar to that found for atmospheric uptake of[¹⁴C]CO₂. Compared with leaves labelled in the light, leaves labelled in the dark had more label in organic acid, amino acid and protein and less label in sugar and starch. After a 5-s pulse the majority of the label fed to petioles in both the light and the dark was found in malate. The majority of the label was found in malate at 120 s in the dark; only 2% of the label was found in phosphorylated compounds at 120 s. The proportion of label found in phosphorylated compounds increased from 17% at 5 s to 80% at 120 s in the light. This suggests that CO₂ delivered to leaves in the light via the transpiration stream is fixed in the veins, a small portion through dark fixation into malate, the remainder by C-3 photosynthesis.     

Boron Deficiency Induced Changes in Translocation of 14CO2-Photosynthate Into Primary Metabolites in Relation to Essential Oil and Curcumin Accumulation in Turmeric (Curcuma longa L.)

Changes in leaf growth, net photosynthetic rate (P _N), incorporation pattern of photosynthetically fixed ¹⁴CO₂ in leaves 1–4 from top, roots, and rhizome, and in essential oil and curcumin contents were studied in turmeric plants grown in nutrient solution at boron (B) concentrations of 0 and 0.5 g m^-3. B deficiency resulted in decrease in leaf area, fresh and dry mass, chlorophyll (Chl) content, and P _N and total ¹⁴CO₂ incorporated at all leaf positions, the maximum effect being in young growing leaves. The incorporation of ¹⁴CO₂ declined with leaf position being maximal in the youngest leaf. B deficiency resulted in reduced accumulation of sugars, amino acids, and organic acids at all leaf positions. Translocation of the metabolites towards rhizome and roots decreased. In rhizome, the amount of amino acids increased but content of organic acids did not show any change, whereas in roots there was decrease in contents of these metabolites as a result of B deficiency. Photoassimilate partitioning to essential oil in leaf and to curcumin in rhizome decreased. Although the curcumin content of rhizome increased due to B deficiency, the overall rhizome yield and curcumin yield decreased. The influence of B deficiency on leaf area, fresh and dry masses, CO₂ exchange rate, oil content, and rhizome and curcumin yields can be ascribed to reduced photosynthate formation and translocation.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

Effect of Zn deficiency on net photosynthetic rate, 14C partitioning,and oil accumulation in leaves of peppermint

Changes in growth, CO₂ exchange rate, and distribution of photosynthetically fixed ¹⁴CO2 into the primary photosynthetic metabolic pool (sugars, amino acids and organic acids) and essential oil accumulation were determined in leaves (leaf positions 1-6 from apex) of developing peppermint grown in a solution culture at Zn concentrations of 0 and 0.05 g m-3. There was a significant decrease in ¹⁴C incorporation in total, ethanol-soluble and ethanol-insoluble fractions in Zn deficient plants at all leaf positions. 14C incorporated in essential oil and in sugars were significantly higher in leaf pairs 1 to 3 than in leaf pairs 4 to 6. ¹⁴C incorporation into amino acids and organic acids was higher in all leaf pairs in Zn deficient plants. Statistical analysis showed a positive significant association between Zn content of leaf and ¹⁴C incorporation into ethanol-soluble fraction and sugars and a negative correlation with ¹⁴C incorporation into amino acids and organic acids. Hence the content of sugars in leaves significantly influences essential oil accumulation under Zn stress. This revised version was published online in September 2006 with corrections to the Cover Date.     

Variation with age in the photosynthetic carbon fixation pattern by leaves of Amaranthus paniculatus and Oryza sativa: Change in the primary carboxylation but no shift from C4 or C3 pathway

Abstract The pattern of photosynthetic carbon fixation by leaves of Amaranthus paniculatus L. (a C₄ plant) and Oryza sativa L. (a C₃ plant) varied with age. Younger leaves of A. paniculatus incorporated ¹⁴CO₂ into malate and aspartate while senescent leaves fixed predominantly into phosphoglycerate (PGA) and sugar phosphates. Only developing leaves of O. sativa formed malate/aspartate whereas mature and senescent leaves produced PGA/sugar phosphates as the initial labelled products. Correspondingly the ratio of phosphoenolpyruvate/ribulose bisphosphate (RuBP) carboxylase activities was higher in younger leaves of A. paniculatus and developing leaves of O. sativa than in older leaves. However, pulse chase experiments revealed that the main donors of carbon to end products, irrespective of leaf stage, were C₄ acids and PGA in A. paniculatus and O. sativa respectively. The results suggest that although an apparent change from initial β-carboxylation to RuBP carboxylation occurs during leaf ontogeny in both the plants, the overall leaf photosynthesis remains C₄ or C₃. The high rate of ¹⁴CO₂ incorporation into PGA/sugar phosphates by senescent leaves of A. paniculatus is suggested to be partly due to the increased intercellular spaces in their mesophyll, allowing greater access of CO₂ directly to RuBP carboxylase in the bundle sheath.     

Effect of Abscisic Acid on the Activities of Photosynthetic Enzymes and 14CO2 Fixation Products in Leaves of Pennisetum typhoides Seedlings

Abscisic acid inhibited the rate of ¹⁴CO₂ fixation in leaves of Pennisetum typhoides (Burm. f.) Stapf & Hubbard seedlings, but increased the activities of phosphoenol-pyruvate-carboxylase and malic enzyme. The leaves of the seedlings grown in the presence of abscisic acid incorporated, in comparison to the control, more radioactivity in the fraction of organic acids, but less radioactivity was recorded in the amino acid fraction. On the other hand, gibberellic acid which also inhibits photosynthetic ¹⁴CO₂ assimilation and decreases the activities of photosynthetic enzymes, favours greater incorporation in alanine, and reduces that in malate. It is deduced that bio-regulants can greatly influence the flow of ¹⁴C into individual photosynthetic products. As in growth, abscisic and gibberellic acids in combination tended to antagonize each other in their effects on enzyme activity as well as in incorporation of ¹⁴CO₂ into photosynthetic products.     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Effect of Oxygen Concentration on C-Photoassimilate Transport from Leaves of Salvia splendens L

Partitioning and transport of recently fixed photosynthate was examined following ¹⁴CO₂ pulse-labeling of intact, attached leaves of Salvia splendens L. maintained in an atmosphere of 300 microliters per liter CO₂ and 20, 210, or 500 milliliters per liter O₂. Under conditions of increasing O₂ (210, 500 milliliters per liter), a smaller percentage of the recently fixed ¹⁴C in the leaf was allocated to starch, whereas a greater percentage of the fixed ¹⁴C appeared in amino acids, particularly serine. The increase in ¹⁴C in amino acids was reflected in material exported from source leaves. A higher percentage of ¹⁴C in serine, glycine, and glutamate was recovered in petiole extracts when source leaves were maintained under elevated O₂ levels. Although pool sizes of these amino acids were increased in both the leaves and petioles with increasing photorespiratory activity, no significant changes in either ¹⁴C distribution or concentration of transport sugars (i.e. stachyose, sucrose, verbascose) were observed. The data indicate that, in addition to being recycled intracellularly into Calvin cycle intermediates, amino acids produced during photorespiration may also serve as transport metabolites, allowing the mobilization of both carbon and nitrogen from the leaf under conditions of limited photosynthesis.     

The Influence of Leaf Age on C(4) Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C(4) Dicot

Characteristics of C₄ photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C₄ dicot). The turnover of [4-¹⁴C] (malate plus aspartate) following a pulse with ¹⁴CO₂ was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of ¹⁴CO₂ incorporation in mid-age leaves was about 1.5-fold higher than in young leaves, and about 2.5-fold higher than in mature leaves. The rate of ¹⁴CO₂ fixation was proportional to the total active pool of malate plus aspartate but was not correlated with the total photosynthetically derived inorganic carbon pool. The leaf's ability to concentrate inorganic carbon photosynthetically declined during leaf expansion, from 29 down to 7 nanomoles per milligram chlorophyll. Similarly, the active aspartate pool also declined during leaf expansion, from about 123 down to 20 nanomoles per milligram chlorophyll. Enhanced metabolism of aspartate to CO₂ and pyruvate in young leaves is suggested to facilitate the maintenance of high CO₂ levels in bundle sheath cells which are thought to have a higher conductance to CO₂.     

Influence of Etherel and Gibberellic Acid on Carbon Metabolism, Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Changes in growth parameters and ¹⁴CO₂ and [U-¹⁴C]-sucrose incorporation into the primary metabolic pools and essential oil were investigated in leaves and stems of M. spicata treated with etherel and gibberellic acid (GA). Compared to the control, GA and etherel treatments induced significant phenotypic changes and a decrease in chlorophyll content, CO₂ exchange rate, and stomatal conductance. Treatment with etherel led to increased total incorporation of ¹⁴CO₂ into the leaves wheras total incorporation from ¹⁴C sucrose was decreased. When ¹⁴CO₂ was fed, the incorporation into the ethanol soluble fraction, sugars, organic acids, and essential oil was significantly higher in etherel treated leaves than in the control. However, [U-¹⁴C]-sucrose feeding led to decreased label incorporation in the ethanol-soluble fraction, sugars, organic acids, and essential oils compared to the control. When ¹⁴CO₂ was fed to GA treated leaves, label incorporation in ethanol-insoluble fraction, sugars, and oils was significantly higher than in the control. In contrast, when [U-¹⁴C]-sucrose was fed the incorporation in the ethanol soluble fraction, sugars, organic acids, and oil was significantly lower than in the control. Hence the hormone treatment induces a differential utilization of precursors for oil biosynthesis and accumulation and differences in partitioning of label between leaf and stem. Etherel and GA influence the partitioning of primary photosynthetic metabolites and thus modify plant growth and essential oil accumulation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

The circadian rhythm of CO₂ assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO₂. During such exposures substantial fixation of CO₂ takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40–50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO₂ to normal air, a circadian rhythm of CO₂ assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO₂, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO₂ in continuous illumination, appears to be due to the presence of a high concentration of CO₂ within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

The incorporation of nitrogen into products of recent photosynthesis in Anabaena cylindrica Lemm.

In vivo tracer studies with ¹⁴C have been performed to help determine pathways of incorporation of newly assimilated nitrogen into N₂-fixing cells of Anabaena cylindrica. After photosynthesis in Ar:O₂:¹⁴CO₂ for 30 min, the addition of N₂ or NH ₄ ⁺ resulted in increased rates of ¹⁴CO₂-incorporation both in the light and dark, and in increased incorporation of ¹⁴C into amino acids at the expense of sucrose and sugar phosphates. Evidence of enhanced sucrose catabolism and increased pyruvate kinase activity was obtained on adding nitrogen, and, of the ¹⁴C-labelling entering the tricarboxylic acid cycle, more appeared in citrate and 2-oxoglutarate than in malate and oxaloacetate. The kinetics of ¹⁴C-incorporation into various amino acids suggest that in the light and dark the most important route of primary ammonia assimilation involves glutamine synthetase and that glutamate, aspartate, glycine and probably alanine are formed secondarily from glutamine.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Incorporation of [14C]Methanol and [14C]Bicarbonate by Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

A study was made of the incorporation of methanol and bicarbonate into the cell constituents of denitrifying or aerobic methanol grown and autotrophic H₂–O₂–CO₂ grown Hyphomicrobium sp. 53-49. Cells were incubated with [¹⁴C]methanol or [¹⁴C]bicarbonate, and the distribution of the radioactivity in the nonvolatile constituents of ethanol extracts of cells was examined. When denitrifying grown cells were incubated with [¹⁴C]methanol, the major part of the radioactivity was fixed to serine as the first stable compound. Aerobic methanol grown cells also fixed [¹⁴C]methanol mainly to serine. These results suggest that methanol grown cells assimilate methanol by the serine pathway. When denitrifying or aerobic methanol grown cells were incubated with [¹⁴C]bicarbonate, malate was mainly observed as a nonvolatile compound in the initial period of the incubation. Autotrophic grown cells also fixed the major part of [¹⁴C]bicarbonate to malate. In this case, phosphoglyceric acid was found in the phosphorylated compounds area.     

Diurnal variations in photosynthetic products and nitrogen metabolism in expanding leaves

Expanding leaves of Capsicum frutescens L. cv. California Wonder, Cucumis melo L. cv. Hales Best, and Citrus sinensis L. Osbeck cv. Washington Navel showed a marked diurnal periodicity in the incorporation of ¹⁴C from photosynthetically fixed ¹⁴CO₂ into amino acids. Incorporation was virtually nil at the beginning of the photoperiod, reached a maximum in the 6th to 7th hour and decreased during the latter part of the photoperiod. In Capsicum frutescens this was apparently a reflection of the availability of reduced nitrogen controlled by the activity of nitrate reductase in the leaves. This also controlled the periodicity of the incorporation of ¹⁴C into fraction I protein. Possible control mechanisms and the relation of nitrogen metabolism to the periodicity of leaf expansion growth are discussed.     

Accumulation of carbon compounds in the epidermis of five species with either different photosynthetic systems or stomatal structure

Abstract Measurements were made of the rate and pattern of ¹⁴CO₂ fixation by the attached and detached epidermis and accompanying leaf tissue of Commelina cyanea (C₃), Zea mays (C₄), Kalanchoë daigremontiana (CAM), Allium cepa (C₃) and Paphiopedilum venustum (C₃). Guard cell plastids of the last two species contain no starch and chlorophyll, respectively. The fine structure of guard cells of these species was also examined. The epidermis of all species when detached from the leaf fixed CO₂ by PEP carboxylation but at rates related to those of the underlying mesophyll. When attached to the leaf during ¹⁴CO₂ feeding a much higher rate of accumulation of radioactivity in substances other than malate and aspartate was found. The results indicated that although concentrations of various metabolites in the two tissues varied greatly, there was fairly ready transport from mesophyll to epidermis. Of recently formed compounds, amino acids appeared to be most readily transported in Commelina and sugars in Zea and Kalanchoë. Epidermal cells appeared to be highly permeable, there being rapid and extensive loss of most labelled products from epidermis placed on CaSO₄ solution.