Peter Dittrich

Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase

The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH2-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.

[14C]Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open

Isolated epidermes of Tulipa gesneriana L. and Commelian communis L. were exposed to 14CO2 in the light and in darkness, when stomata were either closed or open. The labelling patterns did not differ: the main products of CO2 fixation were malate and aspartate. Small amounts of radioactivity appeared also in acids of the tricarboxylic-acid cycle and their transamination products. Since the capacity of epidermis to assimilate CO2 is known to reside in the guard cells, we can state that guard cells continuously take up CO2 if present, and are thus able to recognize the presence of CO2 in their environment at all times. Epidermal samples exposed to 14CO2 in the light contained only small amounts of radioactive 3-phosphoglyceric acid (3-PGA) and sugar phosphates, or none at all. Epidermal samples from Commelina communis did not contain labelled 3-PGA if all adhering mesophyll cells had been removed before exposure to 14CO2. Homogenates of clean epidermal strips of Commelina communis were able to convert exogenous ribulose diphosphate to 3-PGA at a low rate, but could not catalyze the conversion of exogenous ribulose-5-phosphate to ribulose diphosphate. Guard cells of Commelina communis, and probably also those of Tulipa gesneriana, appear not to possess the reductive pentosephosphate pathway, despite the presence of chloroplasts. In such species, the guard cells will have to rely on import in order to maintain their carbon balance. Earlier findings of photosynthetic reduction of CO2 by epidermal tissues were probably obtained with samples that were contaminated with mesophyll cells.

Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning.

Epidermal strips with closed stomata were exposed to malic acid labelled with 14C either uniformly or in 4-C only. During incubation with [U-14C]malate, radioactivity appeared in products of the tricarboxylic-acid cycle and in transamination products within 10 min, in sugars after 2 h. Hardly any radioactivity was found in sugars if [4-14C]malate had been offered. This difference in the degree of labelling of sugars indicates that gluconeogenesis can occur in epidermal tissue, involving the decarboxylation of malate. Epidermis incubated with labelled malate was hydrolyzed after extraction with aqueous ethanol. The hydrolysate contained glucose as the only radioactive product, indicating that starch had been formed from malate. Microautoradiograms were black above stomatal complexes, showing that the latter were sites of starch formation. In order to follow the fate of malate during stomatal closure, malate was labelled in guard cells by exposing epidermes with open stomata to 14CO2 and then initiating stomatal closure. Of the radioactive fixation products of CO2 only malate was released into the water on which the epidermal samples floated; the epidermal strips retained some of the malate and all of its metabolites. In the case of rapid stomatal closure initiated by abscisic acid and completed within 5 min, 63% of the radioactivity was in the malate released, 22% in the malate retained, the remainder in aspartate, glutamate, and citrate. We conclude that during stomatal closing guard cells can dispose of malate by release, gluconeogenesis, and consumption in the tricarboxylic-acid cycle.

Uptake and metabolism of carbohydrates by epidermal tissue

Isolated epidermis of Commelina communis L. and Tulipa gesneriana L. assimilated 14CO2 into malic acid and its metabolites but not into sugars or their phosphates; epidermis could not reduce CO2 by photosynthesis and therefore must be heterotrophic (Raschke and Dittrich, 1977). If, however, isolated epidermis of Commelina communis was placed on prelabelled mesophyll (obtained by an exposure to 14CO2 for 10 min), radioactive sugars appeared in the epidermis, most likely by transfer from the mesophyll. Of the radioactivity in the epidermis, 60% was in sucrose, glucose, fructose, 3-phosphoglyceric acid and sugar phosphates. During a 10-min exposure to 14CO2, epidermis in situ incorporated 16 times more radioactivity than isolated epidermal strips. Isolated epidermis of Commelina communis and Tulipa gesneriana took up 14C-labelled glucose-1-phosphate (without dephosphorylation), glucose, sucrose and maltose. These substances were transformed into other sugars and, simultaneously, into malic acid. Carbons-1 through-3 of malic acid in guard cells can thus be derived from sugars. Radioactivity appeared also in the hydrolysate of the ethanol-insoluble residue and in compounds of the tricarboxylic-acid cycle, including their transamination products. The hydrolysate contained glucose as the only radioactive compound. Radioactivity in the hydrolysate was therefore considered an indication of starch. Starch formation in the epidermis began within 5 min of exposure to glucose-1-phosphate. Autoradiograms of epidermal sections were blackened above the guard cells. Formation of starch from radioactive sugars therefore occurred predominantly in these cells. Epidermis of tulip consistently incorporated more 14C into malic and aspartic acids than that of Commelina communis (e.g. after a 4-h exposure to [14C]glucose in the dark, epidermis, with open stomata, of tulip contained 31% of its radioactivity in malate and aspartate, that of Commelina communis only 2%). The results of our experiments allow a merger of the old observations on the involvement of starch metabolism in stomatal movement with the more recent recognition of ion transfer and acid metabolism as causes of stomatal opening and closing.

Revision of the pathway of D-pinitol formation in Leguminosae

Abstract Tracer studies with labelled inositols demonstrate that in contrast to earlier reports the formation of d -pinitol in Medicago sativa, Ononis spinosa and Trifolium incarnatum does not proceed by epimerization of sequoyitol but via d -ononitol. Hence, the novel pathway detected in Simmondsia chinensis occurs also in the Leguminous family and thus represents a major biosynthetic pathway for d -pinitol.

2-C-methyl-d-erythritol in leaves of Liriodendron tulipifera

Abstract A branched-chain alditol was isolated from yellow autumn leaves of the tulip tree, Liriodendron tulipifera and identified as 2- C -methyl- d -erythritol.

Novel biosynthesis of d-pinitol in simmondsia chinensis

Abstract Chase experiments with 14CO2 and feeding experiments with labelled inositols showed that d -pinitol in leaves of Simmondsia chinensis arises via epimerization of d -ononitol. This finding represents an alternative pathway, since d -pinitol is formed in gymnosperms and other plants by epimerization of sequoyitol.

Inhibition of Stomatal Opening during Uptake of Carbohydrates by Guard Cells in Isolated Epidermal Tissues

The uptake of glucose and other carbohydrates into the guard cells of Commelina communis L. was found to inhibit the opening of the stomata. The concentration of glucose necessary to achieve about 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree of affinity. It is suggested that the uptake of glucose occurs via a proton cotransport, which, depolarizing the membrane potential, slows down the electrogenic import of potassium ions. The process of stomatal closure, in contrast, appears not to be affected by carbohydrate uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, import of glucose or other carbohydrates did not interfere with the regulation of stomatal movements.

Interaction of hydrolytic and phosphorolytic enzymes of starch metabolism in Kalanchoë daigremontiana

The degradation of starch by a protein fraction of Kalanchoë daigremontiana Hamet et Perrier, obtained by ammoniumsulfate precipitation (30–70%), was found to be catalyzed by α-and β-amylase (EC 3.2.1.1 and EC 3.2.1.2, respectively) and by starch phosphorylase (EC 2.4.1.1). The activity of these enzymes was determined by chromatographic analysis of the reaction products; separation and identification of α-amylase was accomplished by heat-inactivation of β-amylase and α-glucosidase. When the interaction of amylolytic and phosphorolytic enzymes was comparatively studied, it was found that without inorganic phosphorus in the reaction mixture, 14C-starch was converted predominantly to maltose and glucose; supplementation with 1–10 mM orthophosphate (Pi) resulted in an increase in glucose-1-phosphate formation and a concomitant reduction of maltose production. Since the total volume of starch degradation remained approximately constant, Pi apparently inhibits β-amylase (Ki about 3 mM Pi). Thus, free Pi in the cell participates in the regulation of starch catabolism, serving as a substrate for starch phosphorylase while simultaneously reducing the production of maltose. With respect to glucan synthesis, adenosinediphosphoglucose-α-1,4-glucosyltransferase (EC 2.4.1.22), maltose phosphorylase and maltoseglucosyltransferase were also found to be active. The last-named enzyme catalyzes an exchange between dextrins and is considered to provide primer carbohydrates for the synthesis of polyglucans.

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

Abstract22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca2+-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.