Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.     

Natural variation in the contribution of microbial density to inducible immune dynamics

Immune responses evolve to balance the benefits of microbial killing against the costs of autoimmunity and energetic resource use. Models that explore the evolution of optimal immune responses generally include a term for constitutive immunity, or the level of immunological investment prior to microbial exposure, and for inducible immunity, or investment in immune function after microbial challenge. However, studies rarely consider the functional form of inducible immune responses with respect to microbial density, despite the theoretical dependence of immune system evolution on microbe‐ versus immune‐mediated damage to the host. In this study, we analyse antimicrobial peptide (AMP) gene expression from seven wild‐caught flour beetle populations (Tribolium spp.) during acute infection with the virulent bacteria Bacillus thuringiensis (Bt) and Photorhabdus luminescens (P.lum) to demonstrate that inducible immune responses mediated by the humoral IMD pathway exhibit natural variation in both microbe density‐dependent and independent temporal dynamics. Beetle populations that exhibited greater AMP expression sensitivity to Bt density were also more likely to die from infection, while populations that exhibited higher microbe density‐independent AMP expression were more likely to survive P. luminescens infection. Reduction in pathway signalling efficiency through RNAi‐mediated knockdown of the imd gene reduced the magnitude of both microbe‐independent and dependent responses and reduced host resistance to Bt growth, but had no net effect on host survival. This study provides a framework for understanding natural variation in the flexibility of investment in inducible immune responses and should inform theory on the contribution of nonequilibrium host‐microbe dynamics to immune system evolution.     

Wheat's wild relatives vary in their response to nitrogen and ozone

The wild relatives of bread wheat ( Triticum aestivum ) are valued by plant breeders for their genetic diversity. However, increasing levels of nitrogen (N) deposition and ground‐level ozone (O₃) threaten plant biodiversity in the Mediterranean and Near‐East, a hotspot for many crop wild relatives. Knowledge of the effect of these air pollutants in combination is still limited, but early indications are that effects vary depending on the level of pollutants, and on the sensitivity of the species to N and O₃. This study examined the responses of four important wheat wild relatives ( Aegilops tauschii , Aegilops speltoides , Triticum dicoccoides and Triticum monococcum ) and one modern wheat cultivar ( T. aestivum ‘Cadenza’) to treatments of N (equivalent to 50 kg ha^?1 year^?1 ammonium nitrate) and O₃ (100 ppb for 21 days), alone and in combination. Measurements included root, shoot and seed biomass, and electrolyte ratios. The O₃ sensitivity of A. tauschii and T. aestivum ‘Cadenza’ were exacerbated by the addition of N, while A. speltoides was found to be nitrophilous, with N ameliorating the negative effect of O_3. Both T. aestivum ‘Cadenza’ and T. dicoccoides produced immature seed heads, with the cultivar's seed head biomass reduced in response to O₃ and N + O₃ while that of T. dicoccoides was largely unaffected. These data suggest that all four wild relatives are likely to be affected when N and O₃ air pollutants co‐occur, and there in situ populations may therefore be at risk. Equally, the results of this study can inform use of their beneficial traits by wheat breeders, and alert them to the inadvertent inclusion of N and O₃ sensitivity.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA‐dependent DEAD‐box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP₆)‐bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure‐function analysis in S. cerevisiae and human (h) cells, and find that the high‐affinity Nup42‐Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy‐terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1‐Dbp5/hDDX19B interaction site. A nup42‐CTD/gle1‐CTD/Dbp5 trimeric complex forms in the presence of IP₆. Deletion of NUP42 abrogates Gle1‐Dbp5 interaction, and disruption of the Nup42 or IP₆ binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42‐CTD and IP₆ stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42‐hGle1B interaction.      

Role of ethylene and related gene expression in the interaction between strawberry plants and the plant growth‐promoting bacterium Azospirillum brasilense

• Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth‐promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum–strawberry plant interaction.
• Ethylene production was evaluated by gas chromatography in plants inoculated or not with A. brasilense REC3. Also, plants were treated with AgNO₃, an inhibitor of ET biosynthesis; with 1‐aminocyclopropane‐1‐carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth‐promoting effect of A. brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT‐PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes.
• Results showed that ET acts as a rapid and transient signal in the first 12 h post‐treatment. A. brasilense REC3‐inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up‐regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A. brasilense REC3 induces production of this auxin, modulating ET signalling.
• Ethylene production and up‐regulation of genes associated with ET signalling in strawberry plants inoculated with A. brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.

     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

Short‐term acclimation to warmer temperatures accelerates leaf carbon exchange processes across plant types

While temperature responses of photosynthesis and plant respiration are known to acclimate over time in many species, few studies have been designed to directly compare process‐level differences in acclimation capacity among plant types. We assessed short‐term (7 day) temperature acclimation of the maximum rate of Rubisco carboxylation (V_cmax), the maximum rate of electron transport (J_max), the maximum rate of phosphoenolpyruvate carboxylase carboxylation (V_pmax), and foliar dark respiration (R_d) in 22 plant species that varied in lifespan (annual and perennial), photosynthetic pathway (C₃ and C₄), and climate of origin (tropical and nontropical) grown under fertilized, well‐watered conditions. In general, acclimation to warmer temperatures increased the rate of each process. The relative increase in different photosynthetic processes varied by plant type, with C₃ species tending to preferentially accelerate CO₂‐limited photosynthetic processes and respiration and C₄ species tending to preferentially accelerate light‐limited photosynthetic processes under warmer conditions. R_d acclimation to warmer temperatures caused a reduction in temperature sensitivity that resulted in slower rates at high leaf temperatures. R_d acclimation was similar across plant types. These results suggest that temperature acclimation of the biochemical processes that underlie plant carbon exchange is common across different plant types, but that acclimation to warmer temperatures tends to have a relatively greater positive effect on the processes most limiting to carbon assimilation, which differ by plant type. The acclimation responses observed here suggest that warmer conditions should lead to increased rates of carbon assimilation when water and nutrients are not limiting.     

The inositol 1,4,5‐trisphosphate receptor is required for maintenance of olfactory adaptation in Drosophila antennae

A role for inositol 1,4,5‐trisphosphate (IP₃) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP₃ signaling pathway, an unequivocal demonstration of IP₃ function in olfaction has not been possible. Here we have investigated the role of a well‐established cellular target of IP₃—the IP₃ receptor (IP₃R)—in olfactory transduction in Drosophila. For this purpose we tested existing viable combinations of IP₃R mutant alleles, as well as a newly generated set of viable itpr alleles, for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP₃R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP₃R. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 282–288, 2000