This research is part of an effort that the ICT (Institute of Tropical Cultivation) has been doing for several years tending to develop superior genotypes of cocoa (Theobroma cacao L.). That is why this study aims to find tolerant or moderately tolerant cocoa genotypes and accessions to water stress with resistance to pests and diseases and high production and industrial quality. Twenty genotypes of cocoa seedlings were investigated, during the period of 6 months, in a soil with sandy-loam texture under nursery conditions, of controlled irrigation. A split plot design was used, with 40 treatments and 3 repetitions. In addition, daily data of the micro climatic characteristics (T °, HR) were taken, in which different indicators of variable were evaluated such as the stomatal conductance (CE) that is greatly influenced by the T ° and HR. The results obtained indicate that the genotypes that showed moderate tolerance to water stress were UNG - 77, UNG - 53, ICT - 1281 and ICT - 1112; the non-tolerant ones were PAS - 93, CEPEC - 2002, ICT - 2142, ICT - 1092, CP - 2005 - C10, TSH - 1188, CCN - 51, IMC - 67, PH - 17, AYP - 15, ICS - 6, BN - 34, ICT - 1506, PAS - 91, PH - 990 and ICS - 1. Under optimal conditions, the most outstanding genotype was ICS-1, both in plant height, number of leaves, and stomatal conductance, this being proof that this genotype develops excellently and stands out if it has the right conditions and water availability.

Stomatal conductance and photosynthesis

For plants, the sensing of light in the environment is as important as vision is for animals. Fluctuations in light can be crucial to competition and survival. One way plants sense light is through the phytochromes, a small family of diverse photochromic protein photoreceptors whose origins have been traced to the photosynthetic prokaryotes. During their evolution, the phytochromes have acquired sophisticated mechanisms to monitor light. Recent advances in understanding the molecular mechanisms of phytochromes and their significance to evolutionary biology make possible an interim synthesis of this rapidly advancing branch of photobiology.

Phytochromes and light signal perception by plants—an emerging synthesis

Previous studies on the Physiology of stomata in higher plants suggest that stomata influence the rate of CO2 fixation in leaf mesophyll tissue. We believe that an equally important stomatal function has not been fully recognised; that stomatal aperture is determined by the capacity of the mesophyll tissue to fix carbon. We altered the capacity of leaves to fix carbon by various means, and found invariably that the diffusive conductance of the epidermis to CO2 transfer, g, (which mainly depends on the number and dimensions of the stomata) changes in nearly the same proportion as the rate of assimilation of CO2. Thus, the intercellular concentration of CO2 (ci), calculated as ci = ca–A/g (where ca is ambient concentration of CO2, A is assimilation rate of CO2), tends to remain constant providing ca is kept constant. We used routine techniques1 to measure A and estimate g in leaves placed singly in chambers. Conductance takes account of CO2 transfer through both stomata and leaf boundary layer, the conductance of the latter being 0.5 mol m−2 s−1.

Stomatal conductance correlates with photosynthetic capacity

Urine has long been a “favored” biofluid among metabolomics researchers. It is sterile, easy-to-obtain in large volumes, largely free from interfering proteins or lipids and chemically complex. However, this chemical complexity has also made urine a particularly difficult substrate to fully understand. As a biological waste material, urine typically contains metabolic breakdown products from a wide range of foods, drinks, drugs, environmental contaminants, endogenous waste metabolites and bacterial by-products. Many of these compounds are poorly characterized and poorly understood. In an effort to improve our understanding of this biofluid we have undertaken a comprehensive, quantitative, metabolome-wide characterization of human urine. This involved both computer-aided literature mining and comprehensive, quantitative experimental assessment/validation. The experimental portion employed NMR spectroscopy, gas chromatography mass spectrometry (GC-MS), direct flow injection mass spectrometry (DFI/LC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) experiments performed on multiple human urine samples. This multi-platform metabolomic analysis allowed us to identify 445 and quantify 378 unique urine metabolites or metabolite species. The different analytical platforms were able to identify (quantify) a total of: 209 (209) by NMR, 179 (85) by GC-MS, 127 (127) by DFI/LC-MS/MS, 40 (40) by ICP-MS and 10 (10) by HPLC. Our use of multiple metabolomics platforms and technologies allowed us to identify several previously unknown urine metabolites and to substantially enhance the level of metabolome coverage. It also allowed us to critically assess the relative strengths and weaknesses of different platforms or technologies. The literature review led to the identification and annotation of another 2206 urinary compounds and was used to help guide the subsequent experimental studies. An online database containing the complete set of 2651 confirmed human urine metabolite species, their structures (3079 in total), concentrations, related literature references and links to their known disease associations are freely available at http://www.urinemetabolome.ca.

/pdf/the-human-urine-metabolome-36so1f5ok7.pdf

The Human Urine Metabolome

The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants†

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.

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

NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoe daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO42−, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn2+, which cannot be replaced by NADP or Mg2+. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn2+, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO2 for recycling during cessation of gas exchange in periods of severe drought.

Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

Epidermal strips with closed stomata were exposed to malic acid labelled with (14)C either uniformly or in 4-C only. During incubation with [U-(14)C]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-(14)C]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 (14)CO2 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.

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

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

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

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.

Peter Dittrich

Papers

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

Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

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

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

Uptake and metabolism of carbohydrates by epidermal tissue.