Seed size,number and strategies in annual plants: a comparative functional analysis and synthesis

Background and AimsPlants depend fundamentally on establishment from seed. However, protocols in trait-based ecology currently estimate seed size but not seed number. This can be rectified. For annuals, seed number should simply be a positive function of vegetative biomass and a negative function of seed size.MethodsUsing published values of comparative seed number as the ‘gold standard’ and a large functional database, comparative seed yield and number per plant and per m² were predicted by multiple regression. Subsequently, ecological variation in each was explored for English and Spanish habitats, newly calculated C-S-R strategies and changed abundance in the British flora.Key ResultsAs predicted, comparative seed mass yield per plant was consistently a positive function of plant size and competitive ability, and largely independent of seed size. Regressions estimating comparative seed number included, additionally, seed size as a negative function. Relationships differed numerically between regions, habitats and C-S-R strategies. Moreover, some species differed in life history over their geographical range. Comparative seed yield per m² was positively correlated with FAO crop yield, and increasing British annuals produced numerous seeds. Nevertheless, predicted values must be viewed as comparative rather than absolute: they varied according to the ‘gold standard’ predictor used. Moreover, regressions estimating comparative seed yield per m² achieved low precision.ConclusionsFor the first time, estimates of comparative seed yield and number for >800 annuals and their predictor equations have been produced and the ecological importance of these regenerative traits has been illustrated. ‘Regenerative trait-based ecology’ remains in its infancy, with work needed on determinate vs. indeterminate flowering (‘bet-hedging’), C-S-R methodologies, phylogeny, comparative seed yield per m² and changing life history. Nevertheless, this has been a positive start and readers are invited to use estimates for >800 annuals, in the Supplementary data, to help advance ‘regenerative trait-based ecology’ to the next level.     

Effects of glutamate, glucose, phosphate, and alkali metal ions on cephamycin C production by Nocardia lactamdurans in defined media

A High cephamycin C producing strain of Nocardia lactam-durans was used to study cell growth and antibiotics production in defined media. Batch fermentations in shake flasks and stirred tanks showed that antibiotic production occurred during cell growth and the production rate rapidly decline as the growth slowed. Glutamate served as a primary substrate during this phase. Later, ammonia was utilized along with a remainder of the glucose. Rapid antibiotic production occurred in this phase. Increased glutamate promoted higher growth, a rise in ammonium ion concentration, and a marked reduction in antibiotic titers. An increase of the glucose concentration along with the glutamate concentration balanced to the medium; no ammonium ion rise occurred and a peak specific antibiotic titer comparable to the control medium was obtained. In a phosphate-limited medium, cell growth equivalent to the control medium and increased antibiotic titers were obtained. In these experiments, adjustment of Na(+) and K(+) ion concentration equal to that in the control medium was found to be important. Based on carbon and nitrogen balances, the activity of the key nitrogen metabolism enzymes, and the published literature, a two-stage model of regulation is suggested.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

Evidence for both humoral and neural regulation of sex pheromone biosynthesis in Spodoptera littoralis

Selected tissues presumably involved in the control of sex pheromone production were analyzed by ELISA for the presence of PBAN-like immunoreactivity (PBAN-IR) in Spodoptera littoralis. The temporal distribution pattern of PBAN-IR in the hemolymph is similar to that of pheromone production in the gland. On the other hand, analysis of the retrocerebral complex, brain-subesophageal ganglion complex, and terminal abdominal ganglion (TAG) revealed similar PBAN-IR levels in both photophase and scotophase periods. Pheromonotropic activity exhibited by both hemolymph and TAG, as determined by a modified in vitro bioassay, agrees with the results of the immunochemical analyses. Severing the ventral nerve cord anterior to the TAG impaired normal sex pheromone production by second-scotophase females. These results are discussed in the context of how sex pheromone biosynthesis is regulated by PBAN in S. littoralis. © 1996 Wiley-Liss, Inc.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts,and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1α-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1α-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1α in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1α showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1α also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1α increased tumor mass by ∼2-fold and tumor volume by ∼3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1α also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1α is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1α expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1α in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1α in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a three-fold reduction in tumor volume. We conclude that HIF1α activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly “feed” cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their “self-digestion.” Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other “classic” oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed “autophagic tumor stroma model of cancer.”Key words: caveolin-1, autophagy, mitophagy, the Warburg effect, tumor stroma, hypoxia, HIF1A, NFκB, compartment-specific oncogenesis, cancer-associated fibroblasts     

Iodination of salicylic acid improves its binding to transthyretin

Transthyretin (TTR) is a plasma homotetrameric protein associated with senile systemic amyloidosis and familial amyloidotic polyneuropathy. In theses cases, TTR dissociation and misfolding induces the formation of amyloidogenic intermediates that assemble into toxic oligomeric species and lead to the formation of fibrils present in amyloid deposits. The four TTR monomers associate around a central hydrophobic channel where two thyroxine molecules can bind simultaneously. In each thyroxine binding site there are three pairs of symmetry related halogen binding pockets which can accommodate the four iodine substituents of thyroxine. A number of structurally diverse small molecules that bind to the TTR channel increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis have been tested. In order to take advantage of the high propensity to interactions between iodine substituents and the TTR channel we have identified two iodinated derivatives of salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid, available commercially. We report in this paper the relative binding affinities of salicylic acid and the two iodinated derivatives and the crystal structure of TTR complexed with 3,5-diiodosalicylic acid, to elucidate the higher binding affinity of this compound towards TTR.     

Thermal clamping of temperature-regulating flowers reveals the precision and limits of the biochemical regulatory mechanism

The flowers of several families of seed plants warm themselves when they bloom. In some species, thermogenesis is regulated, increasing the rate of respiration at lower ambient temperature (T _a) to maintain a somewhat stable floral temperature (T _f). The precision of this regulation is usually measured by plotting T _f over T _a. However, such measurements are influenced by environmental conditions, including wind speed, humidity, radiation, etc. This study eliminates environmental effects by experimentally ‘clamping’ T _f at constant, selected levels and then measuring stabilized respiration rate. Regulating flowers show decreasing respiration with rising T _f (Q ₁₀ < 1). Q ₁₀ therefore becomes a measure of the biochemical ‘precision’ of temperature regulation: lower Q ₁₀ values indicate greater sensitivity of respiration to T _f and a narrower range of regulated temperatures. At the lower end of the regulated range, respiration is maximal, and further decreases in floral temperature cause heat production to diminish. Below a certain tissue temperature (‘switching temperature’), heat loss always exceeds heat production, so thermoregulation becomes impossible. This study compared three species of thermoregulatory flowers with distinct values of precision and switching temperature. Precision was highest in Nelumbo nucifera (Q ₁₀ = 0.16) moderate in Symplocarpus renifolius (Q ₁₀ = 0.48) and low in Dracunculus vulgaris (Q ₁₀ = 0.74). Switching temperatures were approximately 30, 15 and 20°C, respectively. There were no relationships between precision, switching temperature or maximum respiration rate. High precision reveals a powerful inhibitory mechanism that overwhelms the tendency of temperature to increase respiration. Variability in the shape and position of the respiration–temperature curves must be accounted for in any explanation of the control of respiration in thermoregulatory flowers.