Hanna Skubatz

Infrared thermography ofArum lily inflorescences

The infrared radiation emitted from the surface of inflorescences of 12 aroid species was monitored with an infrared camera, capable of 0.1°C resolution, and the data were converted to temperature values by means of temperature reference standards. Images representing surface temperatures were obtained forAmorphophallus bulbifer Blume,A. campanulatus Blume,A. forbesii Engl. et Gehrm.,A. rivieri Dur.,Philodendron selloum Koch,Monstera deliciosa Liebm.,Dracunculus vulgaris Schott,Arum italicum Mill.,A. dioscoridis Sibth.,A. creticum Boiss et Heldr.,Caladium sp., andRemusatia vivipara Schott. These images were different among species with respect to temperature, duration of detectable heat development, and organ type (male and female flowers, spathe and appendix) found to be thermogenic. All these species, however, exhibited three common characteristics: 1) production of heat by the male flowers; 2) pollen-shedding immediately after heat production had ceased; and 3) when male flowers were some distance away from female flowers along the spadix, heat was not detected in female flowers. Heat emission was associated with the alternative, cyanide-insensitive pathway that was fully operative.

Further studies of the glandular tissue of the Sauromatum guttatum (Araceae) appendix.

Electron microscopic studies showed that the trans-Golgi network (trans indicates the polarity of cisternae within the Golgi apparatus; it is opposite to the cis-face that is adjacent to the rough endoplasmic reticulum) was involved in the processing of the osmiophilic material present in the appendix of the inflorescence of Sauromatum guttatum. This material accumulated in the rough endoplasmic reticulum and in special pockets of the plasma membrane prior to heat production. Associations between the endoplasmic reticulum and trans-Golgi network were observed. The Golgi apparatus was composed of 5-6 dictyosomes on one side and one or two somewhat detached cisternae on the other side. Various nonosmiophilic Golgi-derived vesicles were observed: small ones covered with spike-like material, large ones with a smooth surface, and irregularly shaped ones. These electron-translucent vesicles seemed to accumulate in specific localities at the plasma membrane surface in the vicinity of the osmiophilic material; they were not found when the aroma was released. During heat production, the Golgi structures shrank and the activity of the trans-Golgi network seemed to be reduced. At the same time, coated pits were seen at the plasma membrane surface. In some cells, hypertrophic Golgi apparatuses were seen with only 2-3 dictyosomes that contained granulated material in their lumens. Finally, the osmiophilic material was also found in the plasmodesmata.

CYTOCHEMICAL EVIDENCE FOR THE PRESENCE OF ACTIN IN THE NUCLEUS OF THE VOODOO LILY APPENDIX

Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against α-smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti-α-smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC–phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear α-smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43 kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against β-tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti-β-tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50 kDa. This is the first cytochemical evidence for the presence of α-smooth muscle-actin-like protein in the plant nucleus.

Ultrastructural changes in the appendix of the Sauromatum guttatum inflorescence during anthesis

SummaryThe ultrastructure of the epidermal and sub-epidermal cells of the appendix of the Sauromatum guttatum inflorescence reveals developmental changes during anthesis. These changes precede, and probably make possible, heat and odor production. Two days before D-day (the day of heat production and inflorescence-opening) the mitochondria of the epidermis divide; apparent division of the amyloplasts was observed at the same time. The presence of lipid bodies and peroxisomes in the epidermis was clearly evident. On D-day, the epidermis becomes a continuous layer in which the cell walls separating two adjacent cells disappear. At the same time, in the sub-epidermal cells, the mitochondria and the amyloplasts undergo division. The mitochondria become electron-dense, and their DNA is clearly visible. On that day, lipids as well as starch are being depleted. The peroxisomes change in structure every day, from D-2 to D-day. It has also been demonstrated by histochemical techniques that during anthesis the activity of cytochrome c oxidase (3,3-diaminobenzidine as a substrate) decreases whereas the activity of NADH dehydrogenase [tetrazolium salts: nitro-blue tetrazolium chloride (NBT) or neotetrazolium chloride (NT) in the presence of NADH], increases. Oxygen consumption of isolated mitochondria from the D-day appendix was inhibited in the presence of the two tetrazolium salts to a different degree: oxidation of NADH in the presence of NBT was the most sensitive to inhibition, more so than the oxidation of malate and succinate. NT was less effective as an inhibitor in the presence of those three respiratory substrates.

A NAD(P) reductase like protein is the salicylic acid receptor in the appendix of the Sauromatum guttatum inflorescence

The mode of action of the thermogenic inducers (salicylic acid, aspirin, and 2,6-dihydroxybenzoic acid) in the appendix of the Sauromatum guttatum inflorescence is poorly understood. Using ESI-MS and light scattering analysis, we have demonstrated that NAD(P) reductase like protein (RL) is the salicylic acid receptor in the Sauromatum appendix. RL was self-assembled in water into a large unit with a hydrodynamic diameter of 800 nm. In the presence of 1 pM salicylic acid, RL exhibited discontinuous and reversible volume phase transitions. The volume phase changed from 800 to 300 nm diameter and vice versa. RL stayed at each volume phase for ~4–5 min with a fast relaxation time between the 2 phases. ESI-MS analysis of RL extracted from appendices treated with salicylic acid, aspirin, and 2,6-DHBA at a micromolar range demonstrated that these compounds are capable of inducing graded conformational changes that are concentration-dependent. A strong correlation between RL conformations and heat-production induced by salicylic acid was also observed. These preliminary findings reveal structural and conformational roles for RL by which plants regulate their temperature and synchronize their time keeping mechanisms.

Oleic Acid and Its Positional Isomer, cis-Vaccinic Acid, in the Appendix of Sauromatum guttatum during Anthesis

The fatty acid profiles of various organs of the thermogenic inflorescence of Sauromatum guttatum and of the sporophylls of thermogenic male cones of two cycad species (Encephalartos ferox and Dioon edule var edule and var angustifolium) were determined by gas chromatography. During anthesis, palmitate (16:0), oleate [18:1 (9)], cis-vaccinate [18:1 (11)], and linoleate [18:2 (9, 12)] were the most abundant fatty acids in the Sauromatum appendix. cis-Vaccinic acid, a positional isomer of oleic acid, was identified by comparing its retention time on a gas chromatography column and its mass spectrum to an authentic compound. The percentage of oleic acid from total fatty acids dropped from about 9 in the morning 3 d before heat production to 6 in the morning 2 d before heat production. At this time, the percentage of cis-vaccinic acid increased from 3 to 11%, and then remained at this level until the inflorescence dried and died. Palmitoleic acid [16:1 (9)], the common precursor of cis-vaccinic acid, is a minor component of total fatty acids. In six other organs of the Sauromatum inflorescence including thermogenic organs, such as male flowers and lower spadix, palmitate, oleate, and linoleate were prevalent but cis-vaccinate was not. The thermogenic male cones of the two cycad species were rich in palmitic, oleic, and linolenic acids. The level of cis-vaccinic acid in these organs was less than 0.5%.

The energetic state of the thermogenic appendix of the voodoo lily inflorescence. A 31P-NMR study

Abstract Using 31 P-NMR, we have monitored the metabolic changes in the phosphorus compounds of the appendix, the upper thermogenic part of the inflorescence, of Sauromatum guttatum (voodoo lily). Qualitative, in vivo spectra from D− 6 (6 days before heat-production; the day of heat-production is designated D-day). D − 3, D − 2, D − 1. D-day, D + 1 and D + 2, revealed fluctuations in the concentration of the vacuolar and cytoplasmic inorganic phosphate (P i ) relative to an internal standard. From D − 6 to D − 2 the vacuolar P i peak was not prominent relative to the other peaks. In the morning of D − 1, an increase occurred in the vacuolar and the cytoplasmic P i levels. Later on, during development, these levels gradually declined. On D-day, changes in vacuolar and cytoplasmic pH were detected during heat-production. The cytoplasmic pH decreased from 7.7 to 7.2 while that of the vacuole increased by almost one pH unit from 5.8 to 6.7. Perchlorate extract spectra from D − 4, D − 1 and D-day appendices confirmed the observed changes in P i intensity. These spectra also revealed qualitative changes in the content of ATP and ADP. Their sum decreased during development, reaching its lowest level on D-day at the peak of heat-production between 11:00 and 14:00 (as determined by high performance liquid chromatography of the perchlorate extracts). This suggests that in the early morning of D-day, when heat-production starts, around 9:00 phosphofructokinase, the key enzyme in glycolysis, is not activated by a change in the intracellular ATP/ADP ratio, but by other effector(s).

Isolation of mitochondria and mtDNA

The methods described here produce mitochondria with high respiratory activity suitable for the extraction of mitochondrial DNA (mtDNA) of high molecular weight. The mitochondrial preparation involves differential centrifugation and banding in a Percoll gradient [1, 2]. Microbes are washed from the surface of plant tissue with detergent before homogenization. Contaminating nuclear DNA is removed from the mitochondrial preparation by DNase treatment. The procedure has been used with maize, voodoo lily and watermelon yielding mtDNA suitable for restriction analysis.