Cytokinin biochemistry in relation to leaf senescence I. The metabolism of 6-benzylaminopurine and zeatin in oat leaf segments

Journal of Plant Growth Regulation - The metabolism of zeatin and that of 6-benzylaminopurine (BAP) have been compared in oat leaf segments in relation to the markedly differing ability of these...     

Regulators of cell division in plant tissues

[³H]zeatin was supplied through the transpiration stream to de-rooted lupin (Lupinus angustifolius L.) seedlings. The following previously known metabolites were identified chromatographically: 5-phosphates of zeatin riboside and dihydrozeatin riboside, adenosine-5-phosphate, zeatin riboside, zeatin-7-glucopyranoside, zeatin-9-glucopyranoside, adenine, adenosine and dihydrozeatin. Five new metabolites were purified; four of these contain an intact zeatin moiety. Two were identified unequivocally, one as l--[6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purin-9-yl]alanine, a metabolite now termed lupinic acid, and the second as O--d-glucopyranosylzeatin. These two compounds were the major metabolites formed when zeatin solution (100 M) was supplied to the de-rooted seedlings. The radioactivity in the xylem sap of intact seedlings, supplied with [³H]zeatin via the roots, was largely due to zeatin, dihydrozeatin and zeatin riboside. When [³H]zeatin (5 M) was supplied via the transpiration stream to de-rooted Lupinus luteus L. seedlings, the principal metabolite in the lamina was adenosine, while in the stem nucleotides of zeatin and adenine were the dominant metabolites. O-Glucosylzeatin and lupinic acid were also detected as metabolites. The level of the latter varied greatly in the tissues of the shoot, and was greatest in the lower region of the stem and in the expanding lamina. Minor metabolites also detected chromatographically were: (a) dihydrolupinic acid, (b) a partially characterized metabolite which appears to be a 9-substituted adenine (also formed in L. angustifolius), (c) glucosides of zeatin riboside and/or dihydrozeatin riboside, and (d) O-glucosyldihydrozeatin. While lupinic acid supplied exogenously to L. luteus leaves underwent little metabolism, chromatographic studies indicated that O-glucosylzeatin was converted to its riboside, the principal metabolite formed, and also to adenosine, zeatin and dihydrozeatin. A thinlayer chromatography procedure for separating zeatin, dihydrozeatin, zeatin riboside and dihydrozeatin riboside is described.Abbreviations Me₃Si trimethylsilyl - TLC thin-layer chromatography - UV ultraviolet XXIV=Gordon et al., 1975     

Microbial communities and organic biomarkers in a Proterozoic‐analog sinkhole

Little Salt Spring (Sarasota County, FL, USA) is a sinkhole with groundwater vents at ~77 m depth. The entire water column experiences sulfidic (~50 μM) conditions seasonally, resulting in a system poised between oxic and sulfidic conditions. Red pinnacle mats occupy the sediment–water interface in the sunlit upper basin of the sinkhole, and yielded 16S rRNA gene clones affiliated with Cyanobacteria, Chlorobi, and sulfate‐reducing clades of Deltaproteobacteria. Nine bacteriochlorophyll e homologues and isorenieratene indicate contributions from Chlorobi, and abundant chlorophyll a and pheophytin a are consistent with the presence of Cyanobacteria. The red pinnacle mat contains hopanoids, including 2‐methyl structures that have been interpreted as biomarkers for Cyanobacteria. A single sequence of hpnP, the gene required for methylation of hopanoids at the C‐2 position, was recovered in both DNA and cDNA libraries from the red pinnacle mat. The hpnP sequence was most closely related to cyanobacterial hpnP sequences, implying that Cyanobacteria are a source of 2‐methyl hopanoids present in the mat. The mats are capable of light‐dependent primary productivity as evidenced by ¹³C‐bicarbonate photoassimilation. We also observed ¹³C‐bicarbonate photoassimilation in the presence of DCMU, an inhibitor of electron transfer to Photosystem II. Our results indicate that the mats carry out light‐driven primary production in the absence of oxygen production—a mechanism that may have delayed the oxygenation of the Earth's oceans and atmosphere during the Proterozoic Eon. Furthermore, our observations of the production of 2‐methyl hopanoids by Cyanobacteria under conditions of low oxygen and low light are consistent with the recovery of these structures from ancient black shales as well as their paucity in modern marine environments.     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Molecular indicators of microbial diversity in oolitic sands of Highborne Cay,Bahamas

Microbialites (stromatolites and thrombolites) are mineralized mat structures formed via the complex interactions of diverse microbial‐mat communities. At Highborne Cay, in the Bahamas, the carbonate component of these features is mostly comprised of ooids. These are small, spherical to ellipsoidal grains characterized by concentric layers of calcium carbonate and organic matter and these sand‐sized particles are incorporated with the aid of extra‐cellular polymeric substances (EPS), into the matrix of laminated stromatolites and clotted thrombolite mats. Here, we present a comparison of the bacterial diversity within oolitic sand samples and bacterial diversity previously reported in thrombolitic and stromatolitic mats of Highborne Cay based on analysis of clone libraries of small subunit ribosomal RNA gene fragments and lipid biomarkers. The 16S‐rRNA data indicate that the overall bacterial diversity within ooids is comparable to that found within thrombolites and stromatolites of Highborne Cay, and this significant overlap in taxonomic groups suggests that ooid sands may be a source for much of the bacterial diversity found in the local microbialites. Cyanobacteria were the most diverse taxonomic group detected, followed by Alphaproteobacteria, Gammaproteobacteria, Planctomyces, Deltaproteobacteria, and several other groups also found in mat structures. The distributions of intact polar lipids, the fatty acids derived from them, and bacteriohopanepolyols provide broad general support for the bacterial diversity identified through analysis of nucleic acid clone libraries.     

Spatial and temporal variability of biomarkers and microbial diversity reveal metabolic and community flexibility in Streamer Biofilm Communities in the Lower Geyser Basin,Yellowstone National Park

Detailed analysis of 16S rRNA and intact polar lipids (IPLs) from streamer biofilm communities (SBCs), collected from geochemically similar hot springs in the Lower Geyser Basin, Yellowstone National Park, shows good agreement and affirm that IPLs can be used as reliable markers for the microbial constituents of SBCs. Uncultured Crenarchaea are prominent in SBS, and their IPLs contain both glycosidic and mixed glyco‐phospho head groups with tetraether cores, having 0–4 rings. Archaeal IPL contributions increase with increasing temperature and comprise up to one‐fourth of the total IPL inventory at >84 °C. At elevated temperatures, bacterial IPLs contain abundant glycosidic glycerol diether lipids. Diether and diacylglycerol (DAG) lipids with aminopentanetetrol and phosphatidylinositol head groups were identified as lipids diagnostic of Aquificales, while DAG glycolipids and glyco‐phospholipids containing N‐acetylgycosamine as head group were assigned to members of the Thermales. With decreasing temperature and concomitant changes in water chemistry, IPLs typical of phototrophic bacteria, such as mono‐, diglycosyl, and sulfoquinovosyl DAG, which are specific for cyanobacteria, increase in abundance, consistent with genomic data from the same samples. Compound‐specific stable carbon isotope analysis of IPL breakdown products reveals a large isotopic diversity among SBCs in different hot springs. At two of the hot springs, ‘Bison Pool’ and Flat Cone, lipids derived from Aquificales are enriched in ¹³C relative to biomass and approach values close to dissolved inorganic carbon (DIC) (approximately 0‰), consistent with fractionation during autotrophic carbon fixation via the reversed tricarboxylic acid pathway. At a third site, Octopus Spring, the same Aquificales‐diagnostic lipids are 10‰ depleted relative to biomass and resemble stable carbon isotope values of dissolved organic carbon (DOC), indicative of heterotrophy. Other bacterial and archaeal lipids show a similar variance, with values resembling the DIC or DOC pool or a mixture thereof. This variance cannot be explained by hot spring chemistry or temperature alone, but instead, we argue that intermittent input of exogenous organic carbon can result in metabolic shifts of the chemotrophic communities from autotrophy to heterotrophy and vice versa.     

Pathway of malic Acid synthesis in response to ion uptake in wheat and lupin roots: evidence from fixation of C and C

Malate synthesis by CO₂ fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH¹³CO₃ as well as with NaH¹⁴CO₃. The distribution of ¹⁴C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of ¹³C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short-term experiments (15, 30 seconds) showed that ¹⁴C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-¹³C]-, [4-¹³C]-, and [1,4-¹³C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria.