Motif‐based search for a novel fructosyl peptide oxidase from genome databases

The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for α‐glycated compounds, such as HbA1c model compounds fructosyl‐^αN‐valine (f‐^αVal) and fructosyl‐^αN‐valyl‐histidine (f‐^αVal‐His). Unlike previously reported FPOXs, the P. nodorum FPOX has a K_m value for f‐^αVal‐His (0.185 mM) that is considerably lower than that for f‐^αVal (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site‐directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f‐^αVal‐His. A flexible surface loop region was also found to likely play an important role in accepting f‐^αVal‐His. Biotechnol. Bioeng. 2010; 106: 358–366. © 2010 Wiley Periodicals, Inc.     

Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein

Fructosyl peptide oxidases, enzymes that are active against a model compound of glycated hemoglobin, N(alpha)-fructosyl valyl-histidine, were characterized. To identify the primary structure of fructosyl peptide oxidases, we have prepared cDNA libraries from Eupenicillium terrenum ATCC18547 and Coniochaeta sp. NISL9330. The coding regions, both fungal fructosyl peptide oxidases consisting of 1314-bp, were obtained with degenerated primers based on the amino acid sequences and specific primers by 3(') and 5(') RACE (rapid amplification of cDNA ends). By their sequence similarities and substrate specificities, fructosyl peptide oxidases and their homologs could be categorized into two groups: (A) enzymes that preferably oxidize alpha-glycated molecules and (B) enzymes that preferably oxidize epsilon-glycated molecules. We showed that recombinant fructosyl peptide oxidases could be used to detect protease-treated fructosyl-hexapeptide, a glycated peptide that is released from HbA(1C) by endoproteinase Glu-C, suggesting these enzymes could be useful for the enzymatic measurement of HbA(1C).     

C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic pathways and life form types for native species from agro-forestry region,Northeastern China

Of the total 570 species, 194 species in 116 genera and 52 families were found with C₃ photosynthesis, 24 species in 17 genera and 6 families with C₄ photosynthesis, and 2 species in 1 genera and 1 family with CAM photosynthesis. 90 % of the total species can be found in Changbai Mountain flora, more a half (69 %) in North China flora, and about 1/3 in Mongolian flora and Xinan flora, respectively. The occurrence of C₄ species was not as common as that in adjacent grasslands and deserts, but relatively more than in the adjacent forests. Of the total 24 C₄ species, 63 % C₄ species (15 of 24) was found in Gramineae. Nine life form types can be found, reflecting the moist climate in the region, especially the occurrence of epiphyte and liana forms. Relatively more geophyte life form plants suggested the winter in the region was much colder than in grasslands. These indicated that both ecological studies and land management decisions must take into account plant photosynthetic pathway and life form patterns, for both of them are closely related to climatic changes and land use.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

C<Subscript>4</Subscript> grasses in boreal fens: their occurrence in relation to microsite characteristics

C₄ plants are rare in cool climates, an ecological pattern attributable to their poor photosynthetic performance at low temperatures relative to C₃ species. However, some C₄ species are able to persist at high latitudes and high elevations, possibly due to the characteristics of the particular microsites they inhabit in these otherwise unfavourable environments. One such species is Muhlenbergia glomerata, which occurs above 60°N in Canada and is found in the atypical C₄ habitat of boreal fens. In this study, we evaluate how microsite features affect the success of M. glomerata in boreal fens. We surveyed 19 populations across northern Ontario during the summers of 1999 and 2000. The ground coverage by woody vegetation was the most important parameter affecting the presence or absence of M. glomerata. Woody plants covered over 50% of the ground area in plots where M. glomerata is absent, but less than 20% where it is present. The minimum light intensity threshold for the presence of the C₄ species was about 32% of full-sunlight at plant height. Surprisingly, in boreal fens M. glomerata was largely restricted to the wetter moss hollows, rather than occurring on the dry hummocks where its greater water use efficiency might have been advantageous. Woody species dominated the hummocks, but were uncommon in the hollows. In these cool northern climates M. glomerata apparently persists because sufficient periods of temperatures favourable to C₄ photosynthesis occur, but this persistence likely requires some factor that suppresses the woody vegetation.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Long-term effects of elevated atmospheric CO<Subscript>2</Subscript> on species composition and productivity of a southern African C<Subscript>4</Subscript> dominated grassland in the vicinity of a CO<Subscript>2</Subscript> exhalation

We describe the long-term effects of a CO₂ exhalation, created more than 70 years ago, on a natural C₄ dominated sub-tropical grassland in terms of ecosystem structure and functioning. We tested whether long-term CO₂ enrichment changes the competitive balance between plants with C₃ and C₄ photosynthetic pathways and how CO₂ enrichment has affected species composition, plant growth responses, leaf properties and soil nutrient, carbon and water dynamics. Long-term effects of elevated CO₂ on plant community composition and system processes in this sub-tropical grassland indicate very subtle changes in ecosystem functioning and no changes in species composition and dominance which could be ascribed to elevated CO₂ alone. Species compositional data and soil δ¹³C isotopic evidence suggest no detectable effect of CO₂ enrichment on C₃:C₄ plant mixtures and individual species dominance. Contrary to many general predictions C₃ grasses did not become more abundant and C₃ shrubs and trees did not invade the site. No season length stimulation of plant growth was found even after 5 years of exposure to CO₂ concentrations averaging 610 μmol mol⁻¹. Leaf properties such as total N decreased in the C₃ but not C₄ grass under elevated CO₂ while total non-structural carbohydrate accumulation was not affected. Elevated CO₂ possibly lead to increased end-of-season soil water contents and this result agrees with earlier studies despite the topographic water gradient being a confounding problem at our research site. Long-term CO₂ enrichment also had little effect on soil carbon storage with no detectable changes in soil organic matter found. There were indications that potential soil respiration and N mineralization rates could be higher in soils close to the CO₂ source. The conservative response of this grassland suggests that many of the reported effects of elevated CO₂ on similar ecosystems could be short duration experimental artefacts that disappear under long-term elevated CO₂ conditions.     

Effect of fullerenes C<Subscript>60</Subscript> on X-protein amyloids

The inhibitory effect of hydrated fullerene C₆₀ and the sodium salt of the fullerene polycarboxylic derivative C₆₀Cl(C₆H₄CH₂COONa)₅ on the formation of amyloid fibrils by X-protein in vitro has been studied by electron microscopy. It is shown that these compounds not only destroy mature amyloid fibrils but also prevent the formation of new fibrils. This property of fullerenes, which are nanoparticles, can be used to develop a novel medical nanotechnology in the therapy for amyloidoses.     

Solution Structure of a Novel C<Subscript>2</Subscript>-Symmetrical Bifunctional Bicyclic Inhibitor Based on SFTI-1

A novel bifunctional bicyclic inhibitor has been created that combines features both from the Bowman–Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), which has a compact bicyclic structure. The inhibitor was designed by fusing together a pair of reactive loops based on a sequence derived from SFTI-1 to create a backbone-cyclized disulfide-bridged 16-mer peptide. This peptide has two symmetrically spaced trypsin binding sites. Its synthesis and biological activity have been reported in a previous communication [Jaulent and Leatherbarrow, 2004, PEDS 17, 681]. In the present study we have examined the three-dimensional structure of the molecule. We find that the new inhibitor, which has a symmetrical 8-mer half-cystine CTKSIPP′I′ motif repeated through a C₂ symmetry axis also shows a complete symmetry in its three-dimensional structure. Each of the two loops adopts the expected canonical conformation common to all BBIs as well as SFTI-1. We also find that the inhibitor displays a strong and unique structural identity, with a notable lack of minor conformational isomers that characterise most reactive site loop mimics examined to date as well as SFTI-1. This suggests that the presence of the additional cyclic loop acts to restrict conformational mobility and that the deliberate introduction of cyclic symmetry may offer a general route to locking the conformation of β-hairpin structures. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N-fructosyl valyl-histidine (-keto-amine) and N-fructosyl lysine (-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both -keto-amine and -keto-amine, and (2) enzymes that preferably oxidize -keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N-fructosyl valyl-histidine and N-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric (M_r=50,000), was most active at 40 °C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K_m values for N-fructosyl valyl-histidine and N-fructosyl lysine were 2.30 and 1.69 mM, respectively. N-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A_1C, the N-terminal valine residue of the -subunit of which is glycated.Abbreviations HbA_1C Hemoglobin A_1C - FPOX Fructosyl peptide oxidase - FAOX Fructosyl amino acid oxidase - Fru-ValHis N-fructosyl valyl-histidine - Fru-Val N-fructosyl valine - Fru-Lys N-fructosyl lysine - Fru-Gly Fructosyl glycine - TOOS N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt     

Enzymes used for the determination of HbA1C

To develop an enzymatic measurement of HbA(1C), two key enzymes, i.e., fructosyl peptide oxidase and Aspergillus protease were characterized. Fructosyl peptide oxidase from Eupenicillium terrenum was a flavoenzyme that could catalyze the oxidation of N-(1-deoxyfructosyl)-Val-His. The enzyme showed high specificity toward alpha-glycated molecules, therefore it seemed suitable for the HbA(1C) assay. Since high levels of FPOX expression seemed toxic to host cells, we applied a gene expression system using a bacteriophage vector and achieved high levels of expression in Escherichia coli. Next, we found that Aspergillus protease was able to digest N-(1-deoxyfructosyl)-hexapeptide, a glycated peptide that was released from the beta-chain of HbA(1C) by Glu-C endoproteinase. We showed that the N-(1-deoxyfructosyl)-Val-His released from N-(1-deoxyfructosyl)-hexapeptide by Aspergillus protease could be assayed enzymatically using fructosyl peptide oxidase, therefore these enzymes could be applied to the enzymatic measurement of HbA(1C).     

Molecular characteristics of transporters of C<Subscript>4</Subscript>-dicarboxylates and mechanism of translocation

An important role in cell metabolism is played by transport of C₄-dicarboxylates (C₄-DCB). Specifically, they are intermediates of the citrate cycle. Transport of succinate across the mitochondrial membrane provides correlation between metabolism in peroxysomes and in mitochondria. There is known transport of C₄-DCB across all kinds of energy-transforming membranes of the animal, plant, fungal, and bacterial cells. This review summarizes molecular characteristics of the C₄-DCB transporters. Of particular interest are primary structures for the transporters with the known kinetic mechanism and kinetic transport parameters. For each studied group of organisms, the number of transmembrane segments in the carried molecule or the character of specificity does not correlate with the certain transport mechanism—antiport, symport with proton or symport with cation. The review describes perspective methodical approaches allowing association of peculiarities of structure with transport mechanism for individual transporters, preparation of functional hybrid transporters—“protein chimeras,” scanning of transporter transmembrane segments with aid of essential acids, probing of the transporter active center with aid of alkyl and acyl substrate derivatives used to obtain the “lipophilic profile” of the channel of the C₄-DCB transporter. It is recommended to use these approaches to one transporter that has small sizes and large substrate specificity.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Implications of interveinal distance for quantum yield in C<Subscript>4</Subscript> grasses: a modeling and meta-analysis

The distance between veins has the potential to affect photosynthesis in C₄ grasses because photon capture and photosynthetic carbon reduction are primarily restricted to vascular bundle sheath cells (BSC). For example, BSC density should increase as interveinal distance (IVD) decreases, and thus IVD may influence photon capture and photosynthesis in C₄ grasses. The objective of this study is to evaluate the potential importance of IVD to the function of C₄ grasses, and a literature survey is conducted to test the hypothesis that quantum yield of photosynthesis () increases with decreasing IVD. First, a meta-analysis of and IVD values obtained for 12 C₄ grass species supports this hypothesis as and IVD are significantly negatively correlated (r=–0.61). Second, a regression of carbon isotope discrimination () versus IVD was conducted and the regression equation was used in a simple biochemical model that relates to and leakage of CO₂ from the BSC. The modeling analysis also supports the hypothesis that decreases with increasing IVD in C₄ grasses. C₄ grasses are virtually absent from shaded habitats, and the biochemical model is employed to examine the implications of IVD for shade-tolerance in C₄ grasses. The model predicts that only those species with uncommonly small IVD values are able to tolerate prolonged shade.     

Cloning a Truncated Fragment (stx2a<Subscript>1</Subscript>) of the Shiga-Like Toxin 2A<Subscript>1</Subscript> Subunit of EHEC O157:H7: Candidate Immunogen for a Subunit Vaccine

Shiga toxins consist of enzymatically active A and B subunit multimers. The A subunit of shiga-like toxins can be proteolytically cleaved into two parts, A₁ and A₂, with A₁ being responsible for toxic activity. Antibody neutralizing the A₁ subunit of shiga toxin may protect against infection of the enterohemorrhagic Escherichia coli (EHEC O157:H7). It was difficult to express the full-length A₁ subunit of shiga toxin 2 (stx2A₁) in a previous study. We have now analyzed the full-length of stx2A₁ using bioinformatics software. The data show that the carboxyl terminal (of ~15 amino-acid residues) has strong hydrophobicity and low antigenicity. We cloned and expressed a truncated fragment of stx2A₁ (15 amino-acid residues of the carboxyl terminal being removed), designated stx2a₁, which can evoke a humoral immune response. Anti-Stx2a₁ antibodies can neutralize the native shiga toxin 2 both in vivo and in vitro, which suggests that Stx2a₁ serves as a candidate immunogen for a subunit vaccine that can also be used as the antigen to screen phage anti-shiga toxin antibody libraries. L. Liu and H. Zeng contributed equally to this study.     

New Phospholipase A<Subscript>1</Subscript>-producing Bacteria from a Marine Fish

Phospholipase A1 is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of glycerophospholipids to form 2-acyl lysophospholipids. Lysophospholipids are used in foods, cosmetics, and pharmaceuticals as surfactants. Novel forms of phospholipase A1 that function at low temperatures are desirable for use in lipophilic systems in food processing. However, there is currently little variety in the available sources of phospholipase A1. Given this situation, we screened the intestinal contents of marine animals for phospholipase A1-producing bacteria. Colonies that formed a halo on K28CP screening medium and that grew in K28 medium were cultured in liquid K28 medium, and the supernatant was retrieved for analysis. Phosphatidylcholine was added to the culture supernatant, and the product of the reaction was analyzed by using TLC. For culture supernatants that were able to generate lysophosphatidylcholine, synthetic phosphatidylcholines were added, and the site of the reaction was determined by analyzing the fatty acid compositions of the lysophosphatidylcholines generated by GLC. A bacterial isolate from a flatfish, which we named HFKI0020, was found to have phospholipase A1 activity at low temperatures. We determined that the isolate HFKI0020 is closely related to Pseudomonas by using 16S rDNA sequence analysis and by characterizing the isolate with respect to its physiologic and biochemical properties. From the intestinal contents of a marine fish, we successfully isolated a bacterium that secretes phospholipase A1 that is active at low temperatures.     

Light and growth temperature alter carbon isotope discrimination and estimated bundle sheath leakiness in C<Subscript>4</Subscript> grasses and dicots

We combined measurements of short-term (during gas exchange) and long-term (from plant dry matter) carbon isotope discrimination to estimate CO₂ leakiness from bundle sheath cells in six C₄ species (three grasses and three dicots) as a function of leaf insertion level, growth temperature and short-term irradiance. The two methods for determining leakiness yielded similar results (P > 0.05) for all species except Setaria macrostachya, which may be explained by the leaf of this species not being accommodating to gas exchange. Leaf insertion level had no effect on leakiness. At the highest growth temperature (36°C) leakiness was lower than at the two lower growth temperatures (16°C and 26°C), between which no differences in leakiness were apparent. Higher irradiance decreased leakiness in three species, while it had no significant effect on the others (there was an opposite trend in two species). The inverse response to increasing irradiance was most marked in the two NAD-ME dicots (both Amaranthus species), which both showed almost 50% leakiness at low light (300 μmol quanta m⁻² s⁻¹) compared to about 30% at high light (1,600 μmol quanta m⁻² s⁻¹). NADP-ME subtype grasses had lower leakiness than NAD-ME dicots. Although there were exceptions, particularly in the effect of irradiance on leakiness in Sorghum and Boerhavia, we conclude that conditions favourable to C₄ photosynthesis (high temperature and high light) lead to a reduction in leakiness.     

The interactive effects of elevated CO<Subscript>2</Subscript>, temperature and initial size on growth and competition between a native C<Subscript>3</Subscript> and an invasive C<Subscript>3</Subscript> grass

A controlled environment experiment was conducted to determine the impact of enhanced carbon dioxide and temperature on competition between the C₃ grasses Austrodanthonia eriantha and Vulpia myuros. Plants were grown in mixtures and monocultures to compare the responses both with and without an interspecific competitor. Temperature and CO₂ were set at current levels (350 ppm CO₂; 20 °C day and 10 °C night temperature), in factorial combination with enhanced levels (700 ppm CO₂; 23 °C day and 13 °C night temperature). To examine the potential impact of initial seedling size on competition under elevated CO₂ and temperature, the two species were combined in mixtures of differing initial sizes. Above-ground growth of all plants was enhanced by increased CO₂ and temperature alone, however the combined temperature and CO₂ treatment showed a sub-additive effect, where growth was less than expected based on the responses to each factor independently. Austrodanthonia in mixture with Vulpia plants of the same initial size experienced a 27 reduction in growth. Austrodanthonia grown in the presence of an initially larger Vulpia plant experienced a 58 reduction in growth. When the Vulpia plant was initially smaller than Austrodanthonia, growth of the Austrodanthonia was reduced by 16%. The growth of Vulpia appeared to be largely unaffected by the presence of Austrodanthonia. Variation in the CO₂ and temperature environment did not affect the pattern of these interspecific interactions, although there was some evidence to suggest that the degree of suppression of Austrodanthonia by Vulpia was less under elevated CO₂. These results do not support the initial advantage hypothesis, as Vulpia was always able to suppress Austrodanthonia, regardless of the initial relative sizes of the competitors. Furthermore, the lack of an effect of changing the CO₂ or temperature environment on the direction of interspecific competition suggests that the competitiveness of the invasive Vulpia will be minimally affected by changes in atmospheric CO₂ concentration or temperature.