Light-Induced Chloroplast Shrinkage in vivo Detectable After Rapid Isolation of Chloroplasts From Pisum sativum

A light-induced shrinkage of chloroplasts in vivo could be detected with chloroplasts isolated within 2 minutes of harvesting pea plants. As determined both by packed volume and Coulter counter, the mean volume of chloroplasts from plants in the dark was 39 μ³, whereas it was 31 μ³ for chloroplasts from plants in the light. Upon illumination of the plants, the half-time for the chloroplast shrinkage in vivo was about 3 minutes, and the half-time for the reversal in the dark was about 5 minutes. A plant growth temperature of 20° was optimal for the volume change. The chloroplast shrinkage was half-maximal for a light intensity of 400 lux incident on the plants and was light-saturated near 2000 lux. The light-absorbing pigment responsible for the volume change was chlorophyll. This light-induced shrinkage resulted in a flattening and slight indenting of the chloroplasts. This chloroplast flattening upon illumination of the plants may accompany an increase in the photosynthetic efficiency of chloroplasts.     

The relation of light-induced slow absorbancy and scattering changes about 520 nm and structure of chloroplast thylakoids—A theoretical investigation

A theoretical analysis is made on the relation between light-induced thylakoid shrinkage, slow light-induced absorbancy changes about 520 nm, and light-induced scattering changes observed at 90°, which occur in isolated chloroplasts. A simple model of the thylakoids stacks (grana) is assumed and by a mathematical formalism a correlation of these effects is shown. The light minus dark difference spectrum is shown to peak around 520 nm, a fact that confirms earlier suggestions that this difference band is due to the combined effects of the selective dispersion and optical-conformational changes in the grana.     

LIGHT-INDUCED VOLUME CHANGES IN SPINACH CHLOROPLASTS

A light-dependent mechanism that results in a slow, high-amplitude swelling of spinach chloroplasts in vitro has been discovered. The swelling is readily observed by optical and gravimetric methods, and by the use of an electronic particle counter; all show a 100 per cent increase of chloroplast volume in the light with an approximately 10-minute half-time. The existence of an osmotic mechanism for chloroplast swelling in the dark is confirmed. The volume of illuminated chloroplasts versus NaCl concentration represents the addition of osmotic and light effects. The action of light is enhanced by electron flow cofactors, such as phenazine methosulfate (PMS). However, neither conditions for ATP hydrolysis or synthesis nor NH₄Cl influence the time course and extent of swelling. Hence, high-amplitude chloroplast swelling is light- (or electron flow), but not energy-dependent. A remarkable inhibitory effect of inorganic phosphate on chloroplast swelling is observed in the light, but not in the dark. Another action of light on chloroplasts is known to result in a shrinkage of chloroplasts which is rapid, reversible, energy-dependent, and requires phosphate. Thus phosphate determines the action of light on chloroplast volume. Since shrinkage is reversible, but swelling is not, it may be that they reflect physiological and deteriorative processes, respectively. Chloroplasts and mitochondria appear to control their volume by similar mechanisms.     

LIGHT-INDUCED HIGH-AMPLITUDE SWELLING AND SHRINKING IN ISOLATED SPINACH CHLOROPLASTS

It was found that light accelerates the swelling of spinachchloroplasts suspended in a solution containing NaCl and Tris-HCl,and that, in about 60 min, the swollen chloroplasts begin toshrink rapidly. The volume of chloroplasts reached, on swelling,270 per cent of the original volume on the average and thenshrank to 17 per cent. The rate of swelling and the maximumvolume were affected markedly by the tonicity of NaCl. NH₄Cldid not affect the volume changes, while phenazine methosulfateaccelerated the swelling remarkably. The relationship betweenphotoshrinkage and this light-induced high-amplitude swellingwas discussed. (Received April 2, 1966; )     

Energetic basis of the light-induced chloroplast shrinkage in vivo

A light-induced chloroplast shrinkage occurring in vivo wasmeasured with a Coulter counter and a packed weight techniqueusing chloroplasts isolated within two minutes of harvestingpea plants. Introduction of the photosynthetic inhibitor DCMU(5 µM) into the plant either by bathing the cut stem orinjection through a fine hypodermic needle decreased the light-inducedchloroplast shrinkage in vivo 11 to 20%. The uncoupler tri-Fl-CCP(5 µM) inhibited the light-induced shrinkage 80 % , whilenigericin (0.5 µM) completely abolished it. An actionspectrum for the chloroplast volume decrease in vivo had a shoulderat 700-715 mµ. These results are considered in terms ofthe various forms of energy available during photosynthesis.A consistent interpretation is that the lightinduced chloroplastshrinkage in vivo depends either on a high energy state createdby electron flow or on ATP. This chloroplast volume change uponillumination of the plants may increase the photosynthetic efficiency. (Received April 15, 1968; )     

Ultrastructural and Photometric Evidence for Light-Induced Changes in Chloroplast Structure in vivo

Photometric evidence for a reversible, red-light induced transmission decrease in excised leaf tissue or the thalli of certain marine algae has been obtained under conditions which correspond to the occurrence of a light-induced shrinkage of chloroplasts within the cells. Evidence supporting this conclusion is: A) The kinetics of the nonspecific transmission changes are similar to those observed in chloroplasts in vitro. B) The magnitude of the response is larger than could be accounted for by any known pigment which absorbs at 546 mμ. C) The light-induced transmission changes are optimal at pH 5.5 to 6.5 in the presence of electron flow cofactors and weak acid anions, conditions which are optimal for light-induced chloroplast shrinkage in isolated chloroplasts. D) Examination of chloroplast ultrastructure in dark incubated and illuminated chloroplasts reveals a flattening of the chloroplast structure and shrinkage.     

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.Abbreviations EDTA ethylenediamine tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume

Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, −1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at −1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, −2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to −1.8 megapascals and the chloroplasts had higher rates of CO₂-dependent O₂ evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.

Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20°C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20°C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20°C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [¹⁴C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [¹⁴C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.

It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology.

     

Preparation and characterization of phospholipid-depleted chloroplasts

Spinach class II chloroplasts were treated with snake venom phospholipase A2 in the presence of bovine serum albumin, and separated by sucrose-density centrifugation. The treatment yielded phospholipid-depleted chloroplasts which had lost 82.6% of the original phospholipids. About 20% of the phospholipids of chloroplasts were resistant to enzyme attack. These results suggest that phospholipids exist in two states in chloroplast membranes. In spite of considerable phospholipid depletion, the chloroplast preparations retained a large portion of their photoactivities, i.e. light-induced electron transport, light-induced H+ uptake, and light-induced shrinkage. However, cyclic photophosphorylation was significantly affected with the phospholipid removal.     

Phenylmereuric Acetate and Phosphate Control of Light-Dependent Shrinkage and Reactions in Chloroplasts

An investigation of the action of phenylmereuric acetate (PMA) and phosphate on light-induced shrinkage (measured by light scattering and Coulter Counter techniques) and on photosynthetic reactions in spinach chloroplasts led to the following conclusions:

• 1) PMA stimulated light-induced shrinkage (under conditions of cyclic and non-cyclic electron flow) at concentrations which completely inhibited cyclic and non-cyclic photophosphorylation and nicotinamide adenine dinucleotide phosphate (NADP) reduction, though ferricyanide reduction was activated. Although PMA inhibited NADP reduction (probably because this sulfhydryl reagent interfered with the ferredoxin-NADP rednetase) it ean also be considered an uncoiipler (when ferricyanide is the electron acceptor).
• 2) Phosphate maximized light-induced shrinkage (under conditions of cyclic and non cyclic electron flow) at concentrations which did not affect ferricyanide reduction but caused a 40 to 50 per cent inhibition of NADP reduction.
• 3) The pattern of the light scattering response to these two compounds was quite different. In the presence of PMA, the forward (light on) and hack (light off) reactions went to completion rapidly. In the presence of phosphate, the back reaction was rapid but, in the light-induced reaction, three phases were discernible.
• 4) Compared with uncouplers such as NH₄Cl, carbonyl cyanide m-chlorophenyl-hydrazone, pentachlorophenol, and dicoumarol, all of which inhibited both photophosphorylation and conformational changes in chloroplasts, PMA (like quinacrine) had a specific action since it inhibited photophosphorylation while shrinkage was stimulated.
• 5) It appeared that PMA acted at a site beyond the formation of high energy inter-mediates and that, in the absence of photophosphorylation, more energy was diverted to mechanical work (shrinkage). It would seem that, in a cyclic electron flow system, in which ATP synthesis is blocked at a late step (e.g. by PMA), shrinkage may be an indirect method for measuring electron flow.

     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.     

ACTION OF TRITON X-100 ON CHLOROPLAST MEMBRANES : Mechanisms of Structural and Functional Disruption

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100–200 µM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.     

Improved rates of CO2-fixation by intact chloroplasts isolated in media with KCl as the osmoticum

Intact chloroplasts were isolated from spinach leaves using media with either 330 mM sorbitol or 200 mM KCl as the osmoticum. Chloroplasts isolated in KCl exhibited higher rates of CO₂-dependent oxygen evolution in nine out of ten experiments, the average increase being 43%. Chloroplasts isolated in KCl routinely achieved rates of CO₂-dependent oxygen evolution of 200–300 mol·mg chlorophyll^-1·hour^-1 at 20°C. Intact chloroplasts were also isolated in media with 200 mM NaCl or choline chloride but the rates of CO₂ fixation were not superior to those isolated in sorbitol media. The K⁺ content of chloroplasts isolated in KCl media was higher than for chloroplasts isolated in sorbitol. It is suggested that the use of KCl as an osmoticum prevents the loss of chloroplast K⁺ which can occur during isolation in sorbitol media. Chloroplasts isolated in KCl lost, on average, 36% of the initial CO₂ fixation activity after storage for four hours on ice, compared to 24% loss of activity for chloroplasts isolated in sorbitol. This increased loss of activity was not observed if KCl was used in the grinding medium and sorbitol or glycinebetaine in the resuspension media. For measurement of the maximum photosynthetic capacity in vitro, the use of KCl in the grinding medium may be better than sorbitol.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - EDTA ethlenediamine tetraacetic acid     

Cation-induced inhibition of the 515-nm absorption change in chloroplasts: relation to structural changes

Divalent cations were found to inhibit the light-induced 515-nm absorption change in chloroplasts with half-maximal effects occurring between 0.3 and 0.7 mm. Monovalent cations were also effective but higher concentrations (~ 30–40 mm) were required for half-maximal effects. Divalent and monovalent cations also caused absorption changes of chloroplasts in the dark which superficially resemble 515-nm absorption changes. However, they can be correlated with volume changes and represent a combination of turbidity and pigment-absorption changes (flattening) which result from shrinkage. Half-maximal effects occurred at 0.8–1.2 mm for divalent cations and between 15 and 20 mm for monovalent cations. The relationship between salt-induced and osmotic-induced structural changes is also discussed.     

Effect of salts and electron transport on the conformation of isolated chloroplasts. I. Light-scattering and volume changes

Whole chloroplasts isolated from the leaves of spinach (Spinacia oleracea L.) exhibit 2 types of conformational change during electron transport. Amine-uncoupled chloroplasts swell and atebrin-uncoupled chloroplasts shrink. Chloroplasts uncoupled by carbonylcyanide phenylhydrazones and by treatment with ethylenediamine tetraacetic acid do not change their volumes or light-scattering properties during electron transport. Phosphorylating chloroplasts shrink only slightly.The rate and extent of the conformational change parallel the rate of electron transport; both the decrease in turbidity with methylamine and the increase in turbidity with atebrin are rougly proportional to the Hill reaction rate. Consequently the great volume and light-scattering changes which occur in the presence of these uncouplers can be attributed, in part, to the very high rates of uncoupled electron transport. However, for a given rate of electron transport the atebrin-induced scattering increase is very much greater than the increase observed during photophosphorylation.When uncouplers are combined, the carbonylcyanide phenylhydrazone effect (no change) supercedes both the methylamine effect (swelling) and the atebrin effect (shrinking). The methylamine effect supercedes the atebrin (shrinking) and ethylenediamine tetracetic acid (no change) effects. The atebrin effect supercedes the ethylenediamine tetraacetic acid effect. A similar hierarchy of effects is observed with regard to the rate of the uncoupled electron transport.These light-scattering changes of whole chloroplasts reflect similar changes which occur in very small digitonin particles of chloroplasts. Therefore one must look among chloroplast substructures for the basic mechanism of swelling and shrinking.Many salts (including methylamine hydrochloride) cause the chloroplasts to shrink. This phenomenon is not osmotic since comparable osmolarities of sucrose are without effect. Magnesium chloride and calcium chloride are most effective but all salts tested gave major volume decrease when less than 0.05 m. The salt-shrunken chloroplasts show greater light-scattering changes during electron transport than do low-salt chloroplasts.     

Investigations on heat resistance of spinach leaves

Exposure of spinach plants to high temperature (35° C) increased the heat resistance of the leaves by about 3° C. This hardening process occurred within 4 to 6 h, whereas dehardening at 20°/15° C required 1 to 2 days. At 5° C dehardening did not take place. Hardening and dehardening occurred in both the dark and the light. The hardiness was tested by exposure of the leaves to heat stress and subsequent measurements of chlorophyll fluorescence induction and light-induced absorbance changes at 535 nm on the leaves and of the photosynthetic electron transport in thylakoids isolated after heat treatment. Heat-induced damage to both heat-hardened and non-hardened leaves seemed to consist primarily in a breakdown of the membrane potential of the thylakoids accompanied by partial inactivation of electron transport through photosystem II. The increase in heat resistance was not due to temperature-induced changes in lipid content and fatty acid composition of the thylakoids, and no conspicuous changes in the polypeptide composition of the membranes were observed. Prolonged heat treatment at 35° C up to 3 days significantly decreased the total lipid content and the degree of unsaturation of the fatty acids of membrane lipids without further increase in the thermostability of the leaves. Intact chloroplasts isolated from heat-hardened leaves retained increased heat resistance. When the stroma of the chloroplasts was removed, the thermostability of the thylakoids was decreased and was comparable to the heat resistance of chloroplast membranes obtained from non-hardened control plants. Compartmentation studies demonstrated that the content of soluble sugars within the chloroplasts and the whole leaf tissue decreased as heat hardiness increased. This indicated that in spinach leaves, sugars play no protective role in heat hardiness. The results suggest that changes in the ultrastructure of thylakoids in connection with a stabilizing effect of soluble non-sugar stroma compounds are responsible for acclimatization of the photosynthetic apparatus to high temperature conditions. Changes in the chemical composition of the chloroplast membranes did not appear to play a role in the acclimatization.Abbreviations DGDG digalactosyl diglyceride - MGDG monogalactosyl diglyceride - PG phosphatidyl glycerol - PGA 3-phosphoglyceric acid Dedicated to Professor Wilhelm Simonis, Würzburg, on the occasion of his 70th birthday     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Volume-activated Na/H exchange activity in fetal and adult pig red cells: Inhibition by cyclic AMP

Summary Hyposmotic swelling of pig red cells leads to a selective increase in K permeability, whereas hyperosmotic cell shrinkage augments the Na permeability. In this regard, the ouabain-resistant (OR) Na flux of red cells of newborn and adult pigs is characterized in detail. A reduction in cell volume by approximately 18% leads to an increase in the OR Na efflux of fetal and adult cells by 15-and fourfold, respectively. The OR Na influx in both cell types is equally influenced by cell shrinkage. Depletion of cellular K does not influence the volume-activated OR Na efflux. Nor does OR Na influx require external K. Both OR Na efflux and influx activated by shrinkage are inhibited by the diuretics furosemide and amiloride. The rank order of decreasing anion sensitivity for diuretic-sensitive Na efflux was acetate > chloride > gluconate > nitrate. Cell shrinkage induced by the addition of hypertonic salts results in an acidification of the unbuffered and CO₂-free media, provided that both Na and DIDS are present. The qcidification process can be reversed by either of the diuretic agents. These findings suggest that the shrinkageactivated OR Na flux is primarily mediated by a Na/H exchanger rather than by a Na/K/Cl cotransporter. Once loaded with either cAMP or cGMP, cell swelling can no longer activate the Na/H exchanger. The Na/H exchanger activity is detectable in the fetal cells of normal volume but quiescent in adult cells, indicating that the exchanger undergoes a developmental change during the transition from the fetal to adult stage.     

The Effects of Salinity upon Cellular Volume of the Marine Red Alga Porphyra purpurea (Roth) C.Ag.

Changes in cell volume of the marine red alga Porphyra purpureahave been investigated using photomicroscopic and radioisotopictechniques. There is an inverse relationship between cell volumeand external salt content. The alga responds to changes in thewater potential of its bathing medium by rapid swelling in hyposalinemedia and shrinkage in hypersaline conditions. Cells P. purpureabehave as osmometers in concentrated sea-waters, obeying theBoyle-Van't Hoff law. A non-osmotic volume, 20–25% ofthe total cell volume in sea-water, can be predicted from thelinear plot of volume versus reciprocal pressure in concentratedsea-water media. In dilute sea-waters the presence of non-rigidcell walls serves to limit any increases in cell volume. Theprimary response to dilution stress is thus an increase in turgor.Cell volume is not returned to its original value followingprolonged immersion in either hyposaline or hypersaline media,showing that the alga does not ‘osmoregulate’ sensustricto.     

Penitrem and Thomitrem Formation by Penicillium crustosum

The levels penitrem A, B, C, D, E, F, roquefortine C and thomitrem A and Erecovered from extracts of 36 Norwegian, 2 American and one each of Japanese,German, South African, Danish and Fijian isolates of Penicillum crustosumThom were quantitatively determined using high performance liquid chromatography-mass spectrometry (HPLC-MS). Forty-two of the 44 isolates of penitrem-producing isolates grown on rice, afforded levels of thomitrem A and E comparable to that of penitrem A. Thomitrems A and E were also found, but at lower levels, when cultures were grown onbarley. No thomitrems were found when the isolates were grown on liquid media. Theeffects of time and temperature on mycotoxin formation were studied on rice over a 4week period at 10, 15 and 25 °C, respectively. No mycotoxins could be detectedafter 1 week at 10 °C, but after 2 weeks at 10 °C levels were similar tothose produced at 15 and 25 °C. Higher levels of thomitrems A and E weredetected when media were maintained at lower pH. The possibility that thomitremsA and E might be derived by acid promoted conversion of penitrems A and E wasexplored in stability trials performed at pH 2, 3, 4, 5 and 7 in the presence and absenceof media. Thomitrems were formed at pH 2, 3 and 4 but not at pH 5 and 7.