The nitrate transporter MtNPF6.8 (MtNRT1.3) transports abscisic acid and mediates nitrate regulation of primary root growth in Medicago truncatula.

Elongation of the primary root during postgermination of Medicago truncatula seedlings is a multigenic trait that is responsive to exogenous nitrate. A quantitative genetic approach suggested the involvement of the nitrate transporter MtNPF6.8 (for Medicago truncatula NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER Family6.8) in the inhibition of primary root elongation by high exogenous nitrate. In this study, the inhibitory effect of nitrate on primary root elongation, via inhibition of elongation of root cortical cells, was abolished in npf6.8 knockdown lines. Accordingly, we propose that MtNPF6.8 mediates nitrate inhibitory effects on primary root growth in M. truncatula. pMtNPF6.8:GUS promoter-reporter gene fusion in Agrobacterium rhizogenes-generated transgenic roots showed the expression of MtNPF6.8 in the pericycle region of primary roots and lateral roots, and in lateral root primordia and tips. MtNPF6.8 expression was insensitive to auxin and was stimulated by abscisic acid (ABA), which restored the inhibitory effect of nitrate in npf6.8 knockdown lines. It is then proposed that ABA acts downstream of MtNPF6.8 in this nitrate signaling pathway. Furthermore, MtNPF6.8 was shown to transport ABA in Xenopus spp. oocytes, suggesting an additional role of MtNPF6.8 in ABA root-to-shoot translocation. (15)NO3(-)-influx experiments showed that only the inducible component of the low-affinity transport system was affected in npf6.8 knockdown lines. This indicates that MtNPF6.8 is a major contributor to the inducible component of the low-affinity transport system. The short-term induction by nitrate of the expression of Nitrate Reductase1 (NR1) and NR2 (genes that encode two nitrate reductase isoforms) was greatly reduced in the npf6.8 knockdown lines, supporting a role of MtNPF6.8 in the primary nitrate response in M. truncatula.

Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 µM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 µM and 39.3 µM whereas LMA10203 and zacopride possessed IC50 values of 8.07 µM and 7.04 µM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor.

Ca²âº/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²âº signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula.

Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.

Molecular and functional characterization of a novel sodium channel TipE-like auxiliary subunit from the American cockroach Periplaneta americana.

In Drosophila melanogaster, the functions of voltage-gated sodium (Nav) channels are modulated by TipE and its orthologs. Here, we describe a novel TipE homolog of the American cockroach, Periplaneta americana, called PaTipE. Like DmTipE, PaTipE mRNAs are ubiquitously expressed. Surprisingly, PaTipE mRNA was undetectable in neurosecretory cells identified as dorsal unpaired median neurons. Phylogenetic analysis placed this new sequence in TipE clade, indicating an independent evolution from a common ancestor. Contrary to previous reports, our data indicate that the auxiliary subunits of insect Nav channels are very distant from the mammalian BKCa auxiliary subunits. To decipher the functional roles of PaTipE, we characterized the gating properties of DmNav1-1 channels co-expressed with DmTipE or PaTipE, in Xenopus oocytes. Compared to DmTipE, PaTipE increased Na(+) currents by a 4.2-fold. The voltage-dependence of steady-state fast inactivation of DmNav1-1/PaTipE channels was shifted by 5.8 mV to more negative potentials than that of DmNav1-1/DmTipE channels. DmNav1-1/PaTipE channels recovered 3.2-fold slower from the fast-inactivated state than DmNav1-1/DmTipE channels. In conclusion, this study supports that the insect Nav auxiliary subunits share functional features with their mammalian counterparts, although structurally and phylogenetically distant.

Similar Comparative Low and High Doses of Deltamethrin and Acetamiprid Differently Impair the Retrieval of the Proboscis Extension Reflex in the Forager Honey Bee (Apis mellifera).

In the present study, the effects of low (10 ng/bee) and high (100 ng/bee) doses of acetamiprid and deltamethrin insecticides on multi-trial learning and retrieval were evaluated in the honey bee Apis mellifera. After oral application, acetamiprid and deltamethrin at the concentrations used were not able to impair learning sessions. When the retention tests were performed 1 h, 6 h, and 24 h after learning, we found a significant difference between bees after learning sessions when drugs were applied 24 h before learning. Deltamethrin-treated bees were found to be more sensitive at 10 ng/bee and 100 ng/bee doses compared to acetamiprid-treated bees, only with amounts of 100 ng/bee and at 6 h and 24 h delays. When insecticides were applied during learning sessions, none of the tested insecticides was able to impair learning performance at 10 ng/bee or 100 ng/bee but retention performance was altered 24 h after learning sessions. Acetamiprid was the only one to impair retrieval at 10 ng/bee, whereas at 100 ng/bee an impairment of retrieval was found with both insecticides. The present results therefore suggest that acetamiprid and deltamethrin are able to impair retrieval performance in the honey bee Apis mellifera.

Characterization of tyramine ß-hydroxylase, an enzyme upregulated by stress in Periplaneta americana.

Octopamine (OA) is an important neuroactive substance that modulates several physiological functions and behaviors of various invertebrate species. This biogenic monoamine, structurally related to noradrenaline, acts as a neurotransmitter, a neuromodulator, or a neurohormone in insects. The tyramine ß-hydroxylase (TBH) catalyzes the last step in OA biosynthesis and thus plays a key role in the regulation of synthesis and secretion of OA in neurons. The aim of this study was to characterize TBH in the cockroach Periplaneta americana and to get a better understanding of its regulation under stress conditions in this insect. First of all, five full-length cDNAs encoding TBH isoforms were cloned from the nerve cord of the physiological model P. americana. PaTBH transcripts were found mainly expressed in nervous tissues and in octopaminergic dorsal unpaired median neurons. In addition, a new ELISA assay was developed so as to allow determination of both OA level and TBH activity in stressed cockroaches. Mechanical stressful stimulation led to a significant increase in TBH activity after 1 and 24  h, with a higher induction after 1  h than after 24  h. Thus, TBH could be considered as a promising biomarker of stress in insects rather than OA.

Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.