AC1 Gene Research Articles

In silico identification of papaya genome-encoded microRNAs to target begomovirus genes in papaya leaf curl disease.

Papaya leaf curl disease (PaLCuD) is widespread and classified in the genus begomovirus (Geminiviridae), disseminated by the vector whitefly Bemisia tabaci. RNA interference (RNAi)-based antiviral innate immunity stands as a pivotal defense mechanism and biological process in limiting viral genomes to manage plant diseases. The current study aims to identify and analyze Carica Papaya locus-derived capa-microRNAs with predicted potential for targeting divergent begomovirus species-encoded mRNAs using a 'four integrative in silico algorithms' approach. This research aims to experimentally activate the RNAi catalytic pathway using in silico-predicted endogenous capa-miRNAs and create papaya varieties capable of assessing potential resistance against begomovirus species and monitoring antiviral capabilities. This study identified 48 predicted papaya locus-derived candidates from 23 miRNA families, which were further investigated for targeting begomovirus genes. Premised all the four algorithms combined, capa-miR5021 was the most anticipated miRNA followed by capa-miR482, capa-miR5658, capa-miR530b, capa-miR3441.2, and capa-miR414 'effective' papaya locus-derived candidate capa-miRNA and respected putative binding sites for targets at the consensus nucleotide position. It was predicted to bind and target mostly to AC1 gene of the complementary strand and the AV1 gene of the virion strand of different begomovirus isolates, which were associated with replication-associated protein and encapsidation, respectively, during PaLCuD. These miRNAs were also found targeting betaC1 gene of betasatellite which were associated with retardation in leaf growth and developmental abnormalities with severe symptoms during begomovirus infection. To validate target prediction accuracy, we created an integrated Circos plot for comprehensive visualization of host-virus interaction. In silico-predicted papaya genome-wide miRNA-mediated begomovirus target gene regulatory network corroborated interactions that permit in vivo analysis, which could provide biological material and valuable evidence, leading to the development of begomovirus-resistant papaya plants. The integrative nature of our research positions it at the forefront of efforts to ensure the sustainable cultivation of papaya, particularly in the face of evolving pathogenic threats. As we move forward, the knowledge gained from this study provides a solid foundation for continued exploration and innovation in the field of papaya virology, and to the best of our knowledge, this study represents a groundbreaking endeavor, undertaken for the first time in the context of PaLCuD research.

Research Article A new Informatics Framework for Evaluating the Codon Usage Metrics, Evolutionary Models and Phylogeographic reconstruction of Tomato yellow leaf curl virus (TYLCV) in different regions of Asian countries

: Tomato yellow leaf curl virus (TYLCV) is a major devastating viral disease, majorly affecting the tomato production globally. The disease is majorly transmitted by the Whitefly. The Begomovirus (TYLCV) having a six major protein coding genes, among them the C1/AC1 is evidently associated with viral replication. Owing to immense role of C1/AC1 gene, the present study is an initial effort to elucidate the factors shaping the codon usage bias and evolutionary pattern of TYLCV-C1/AC1 gene in five major Asian countries. Based on publically available nucleotide sequence data the Codon usage pattern, Evolutionary and Phylogeographic reconstruction was carried out. The study revealed the presence of significant variation between the codon bias indices in all the selected regions. Implying that the codon usage pattern indices (eNC, CAI, RCDI, GRAVY, Aromo) are seriously affected by selection and mutational pressure, taking a supremacy in shaping the codon usage bias of viral gene. Further, the tMRCA age was 1853, 1939, 1855, 1944, 1828 for China, India, Iran, Oman and South Korea, respectively for TYLCV-C1/AC1 gene. The integrated analysis of Codon usage bias, Evolutionary rate and Phylogeography analysis in viruses signifies the positive role of selection and mutational pressure among the selected regions for TYLCV (C1/AC1) gene.

Research Article A new Informatics Framework for Evaluating the Codon Usage Metrics, Evolutionary Models and Phylogeographic reconstruction of Tomato yellow leaf curl virus (TYLCV) in different regions of Asian countries

Tomato yellow leaf curl virus (TYLCV) is a major devastating viral disease, majorly affecting the tomato production globally. The disease is majorly transmitted by the Whitefly. The Begomovirus (TYLCV) having a six major protein coding genes, among them the C1/AC1 is evidently associated with viral replication. Owing to immense role of C1/AC1 gene, the present study is an initial effort to elucidate the factors shaping the codon usage bias and evolutionary pattern of TYLCV-C1/AC1 gene in five major Asian countries. Based on publically available nucleotide sequence data the Codon usage pattern, Evolutionary and Phylogeographic reconstruction was carried out. The study revealed the presence of significant variation between the codon bias indices in all the selected regions. Implying that the codon usage pattern indices (eNC, CAI, RCDI, GRAVY, Aromo) are seriously affected by selection and mutational pressure, taking a supremacy in shaping the codon usage bias of viral gene. Further, the tMRCA age was 1853, 1939, 1855, 1944, 1828 for China, India, Iran, Oman and South Korea, respectively for TYLCV-C1/AC1 gene. The integrated analysis of Codon usage bias, Evolutionary rate and Phylogeography analysis in viruses signifies the positive role of selection and mutational pressure among the selected regions for TYLCV (C1/AC1) gene.

Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato.

Expression of artificial microRNA targeting ATP binding domain of AC1 in transgenic tomato confers resistance to Tomato leaf curl disease without impacting the yield of tomato. Tomato curl leaf disease caused by Tomato leaf curl virus (ToLCV) is a key constraint to tomato cultivation worldwide. Engineering transgenic plants expressing artificial microRNAs (amiRNAs) against the AC1 gene of Tomato leaf curl New Delhi virus (ToLCNDV), which is important for virus replication and pathogenicity, would consequently confer virus resistance and reduce crop loss in the economically important crops. This study relates to an amiRNA developed on the sequence of Arabidopsis miRNA319a, targeting the ATP/GTP binding domain of AC1 gene of ToLCNDV. The AC1-amiR was found to regulate the abundance of AC1, providing an excellent strategy in providing defense against ToLCNDV. Transgenic lines over-expressing AC1-amiR, when challenged with ToLCNDV, showed reduced disease symptoms and high percentage resistance ranging between ∼40 and 80%. The yield of transgenic plants was significantly higher upon ToLCNDV infection as compared to the non-transgenic plants. Although the natural resistance resources against ToLCNDV are not available, this work streamlines a novel amiRNA-based mechanism that may have the potential to develop viral resistance strategies in tomato, apart from its normal symptom development properties as it is targeting the conserved region against which higher accumulation of small interfering RNAs (siRNA) occurred in a naturally tolerant tomato cultivar.

RNAi-based transgene conferred extreme resistance to the geminivirus causing apical leaf curl disease in potato

Potato apical leaf curl disease is an emerging geminiviral disease in tropics and subtropics. It was reported for the first time in the year 1999 in northern plains of India but quickly spread to almost all potato growing regions of the country largely due to prevalence of warmer weather during early crop growth, thereby favoring whitefly vector. The problem of apical leaf curl disease in India became more severe due to lack of seed indexing for this virus in conventional seed production scheme. Although it accounts for major yield loss, there is no conventional source of resistance available in potato against Tomato Leaf Curl New Delhi Virus-Potato (ToLCNDV-Potato) that causes this disease in potato. In the present study, we have investigated the potential use of RNAi for obtaining resistance against this DNA virus in potato. The replication-associated protein gene (AC1) of the virus was used to obtain pathogen-derived resistance. The AC1 gene was PCR amplified from field-infected potato leaves, cloned and sequenced (JN393309). It showed 93% sequence similarity with the AC1 gene of Tomato Leaf Curl Virus-New Delhi (TOLCV-NDe; DQ169056) virus. Transgenic plants encoding the AC1 gene in three different orientations, viz. sense, antisense and hairpin loop, were raised. Transgenic lines when challenge inoculated with ToLCNDV-Potato showed different levels of resistance for all three constructs. Transgene integration and copy number in selected transgenic lines were determined by qPCR and further confirmed by Southern blot analysis. Though a reduction in viral titer was observed in transgenic lines encoding either antisense or hairpin loop constructs of AC1 gene, the latter transgenics showed most significant results as shown by reduction in the level of symptom expression in glasshouse screening as well as real-time data of in vivo virus concentration. In fact, we obtained a few totally asymptomatic transgenic lines with hairpin loop strategy.

Enhancing the pathogenicity of Arthrobotrys conoides and A. oligospora against Meloidogyne javanica J2 by transferring of protease (Ac1) gene and evaluation of antagonistic capability of transgenic isolates

Antagonistic fungi are well-known as viable alternatives to chemical control of root-knot nematodes. In this paper, serine protease Ac1 as an important pathogenicity factor was used to enhance the antagonistic activity of Arthrobotrys conoides and A. oligospora against Meloidogyne javanica J2 (second stage juveniles). Ac1 gene was extracted from A. conoides and cloned in pCAMBIA1304 vector. The recombinant plasmid was then transferred to these fungi using two strains of Agrobacterium tumefaciens (LBA4404 and AGL1). Transgenic isolates were confirmed by PCR amplification of Hygromycin B resistance gene (hph) as selectable marker, protease assay using casein substrate and in vitro bioassay. Transferring of Ac1 gene by homologous recombination to A. conoides, increased protease activity. Our results showed that production of protease in both transgenic species was increased compared to wild type. Bioassay results indicated that pathogenicity rates of transformants, in both the number of traps formed and the number of trapped J2, increased compared to the wild type. The results showed that inhibition of root-knot nematode, M. javanica, was markedly increased by transgenic isolates of Arthrobotrys spp. compared to the wild types.

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression.

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

Simultaneous Detection of Both RNA and DNA Viruses Infecting Dry Bean and Occurrence of Mixed Infections by BGYMV, BCMV and BCMNV in the Central-West Region of Mexico.

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed to simultaneously detect bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV), and bean golden yellow mosaic virus (BGYMV) from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB) method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the AC1 gene of BGYMV as well as 469 bp and 746 bp from the CP gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%), 41/187 (21.9%), and 35/187 (18.7%), for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5%) samples were co-infected with two of these viruses and 10/187 (5.3%) with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.

Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.

Ca 2+ -Stimulated Adenylyl Cyclase AC1 Generates Efficient Biological Pacing as Single Gene Therapy and in Combination With HCN2

Biological pacing performed solely via HCN2 gene transfer in vivo results in relatively slow idioventricular rates and only moderate autonomic responsiveness. We induced biological pacing using the Ca(2+)-stimulated adenylyl cyclase AC1 gene expressed alone or in combination with HCN2 and compared outcomes with those with single-gene HCN2 transfer. We implanted adenoviral HCN2, AC1, or HCN2/AC1 constructs into the left bundle branches of atrioventricular-blocked dogs. During steady-state gene expression (days 5-7), differences between AC1, HCN2/AC1, and HCN2 alone were evident in basal beating rate, escape time, and dependence on electronic backup pacing. In HCN2, AC1, and HCN2/AC1, these parameters were as follows: basal beating rate: 50±1.5, 60±5.0, and 129±28.9 bpm (P<0.05 for HCN2/AC1 versus HCN2 or AC1 alone), respectively; escape time: 2.4±0.2, 1.3±0.2, and 1.1±.0.4 seconds (P<0.05 for AC1 and HCN2/AC1 versus HCN2); and percent electronic beats: 34±8%, 2±1%, and 6±2% (P<0.05 for AC1 and HCN2/AC1 versus HCN2). Instantaneous (SD1) and long-term (SD2) heart rate variability and circadian rhythm analyzed via 24-hour Holter recordings showed a shift toward greater sensitivity to parasympathetic modulation in animals injected with AC1 and a high degree of sympathetic modulation in animals injected with HCN2/AC1. AC1 or HCN2/AC1 overexpression in left bundle branches provides highly efficient biological pacing and greater sensitivity to autonomic modulation than HCN2 alone.

Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences

Several important biological processes are performed by distinct functional domains found on replication-associated protein (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal 669 bp (5'AC1) and C-terminal 783 bp (3'AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens to make use of an interference strategy for impairing cotton leaf curl virus (CLCuV) infection in transgenic cotton. Compared with nontransformed control, we observed that transgenic cotton plants overexpressing either N-terminal (5'AC1) or C-terminal (3'AC1) sequences confer resistance to CLCuV by inhibiting replication of viral genomic and β satellite DNA components. Molecular analysis by Northern blot hybridization revealed high transgene expression in early and late growth stages associated with inhibition of CLCuV replication. Of the eight T(1) transgenic lines tested, six had delayed and minor symptoms as compared to nontransformed control lines which developed disease symptoms after 2-3 weeks of whitefly-mediated viral delivery. Virus biological assay and growth of T(2) plants proved that transgenic cotton plants overexpressing 5'- and 3'AC1 displayed high resistance level up to 72, 81%, respectively, as compared to non-transformed control plants following inoculation with viruliferous whiteflies giving significantly high cotton seed yield. Progeny analysis of these plants by polymerase chain reaction (PCR), Southern blotting and virus biological assay showed stable transgene, integration, inheritance and cotton leaf curl disease (CLCuD) resistance in two of the eight transgenic lines having single or two transgene insertions. Transgenic cotton expressing partial AC1 gene of CLCuV can be used as virus resistance source in cotton breeding programs aiming to improve virus resistance in cotton crop.

Complete nucleotide sequence of a monopartite begomovirus associated with yellow vein mosaic disease of mesta from north India

Yellow vein mosaic disease of mesta in northern India was found to be associated with a distinct begomovirus species. Except the AC1 gene, this begomovirus isolate shares low sequence identity with the Mesta yellow vein mosaic virus reported to be associated with a similar disease of mesta from eastern India.

Pathogenicity of a naturally occurring recombinant DNA satellite associated with tomato yellow leaf curl China virus

Recombinant DNA beta molecules (RecDNA-Abeta) comprising parts of DNA A and DNA beta associated with tomato yellow leaf curl China virus (TYLCCNV) have been identified in naturally infected tobacco plants. Several examples of the recombinant DNA have been cloned and characterized by sequence analysis. All are approximately half the size of TYLCCNV genomic DNA, and all contain the betaC1 gene and the A-rich region from TYLCCNV DNA beta as well as intergenic region sequences and the 5' terminus of the AC1 gene from TYLCCNV DNA A. RecDNA-Abeta was detected by PCR in five of 25 TYLCCNV isolates. Co-inoculation of TYLCCNV DNA A and RecDNA-Abeta induced symptoms indistinguishable from those induced by TYLCCNV DNA A and DNA beta in Nicotiana benthamiana, Nicotiana glutinosa, Solanum lycopersicum and Petunia hybrida plants, and Southern blot hybridization results showed that RecDNA-Abeta could replicate stably in N. benthamiana plants.

RNAi-Mediated Resistance to Bean golden mosaic virus in Genetically Engineered Common Bean (Phaseolus vulgaris)

Bean golden mosaic virus (BGMV) is transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner, causing the golden mosaic of common bean (Phaseolus vulgaris L.). The characteristic symptoms are yellow-green mosaic of leaves, stunted growth, or distorted pods. The disease is the largest constraint to bean production in Latin America and causes severe yield losses (40 to 100%). Here, we explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generate highly resistant transgenic common bean plants. Eighteen transgenic common bean lines were obtained with an intron-hairpin construction to induce post-transcriptional gene silencing against the AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of the plants were free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant during the whole plant life cycle) and at a very early stage of plant development. Transgene-specific small interfering RNAs were detected in both inoculated and non-inoculated transgenic plants. A semiquantitative polymerase chain reaction analysis revealed the presence of viral DNA in transgenic plants exposed to viruliferous whiteflies for a period of 6 days. However, when insects were removed, no virus DNA could be detected after an additional period of 6 days.

Studies on the activity of a bidirectional promoter of Mungbean yellow mosaic India virus by agroinfiltration

The AV promoter expressing AV1 and AV2 genes and AC1 promoter expressing AC1 gene are present in opposite orientation in the intergenic region of Mungbean yellow mosaic India virus (MYMIV). Transient Agrobacterium-mediated delivery of putative promoter constructs into Nicotiana benthamiana and different legumes, followed by reporter gene (β- d-glucuronidase, GUS) assay, identified the promoter region of both AC1 and AV genes that is necessary for transcriptional initiation. Transcription activator protein-independent activity of AV promoter and differential regulation of AC1 promoter are unique to MYMIV. The AV promoter is a composite core promoter having both TATA box and Initiator elements (TATA +Inr +). Many transcription factor binding sites were identified in the upstream promoter sequences of both virion and complementary sense genes, which might be used in the transcription regulation studies of the host plant as well as the virus.

Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

Spatiotemporal localization of the calcium-stimulated adenylate cyclases, AC1 and AC8, during mouse brain development

Type 1 and type 8 adenylate cyclases, AC1 and AC8, are membrane bound enzymes that produce cAMP in response to calcium entry and could thus control a large number of developmental processes. We provide a detailed spatiotemporal localization of these genes in the mouse brain during embryonic and postnatal life using in situ hybridization. AC1 gene expression begins early in embryonic life (before E13), and its expression is much more widespread than in adults. Transient expression of AC1 is found in the striatum, the dorsal thalamus, the trigeminal nerve nuclei, the Purkinje cells of the cerebellum, the interneurons of the hippocampus, and the retinal ganglion cells. In all these structures, the peak of AC1 gene expression occurs during early postnatal life, decreasing by P10. After P15, AC1 expression is confined to the hippocampus, the cerebral cortex, and to the granule cells of the cerebellum. AC8 gene expression also begins early in embryonic life (E12)--but in a more limited number of regions than in adults. AC8 expression is initially restricted to the epithalamus, the hypothalamus, the superior colliculus, the cerebellar anlage the proliferative zone of the rhombic lip, and the spinal cord. The expression increases and broadens during postnatal life, particularly in the thalamus and the cerebral cortex. A transient peak of AC8 expression is found in layer IV of the somatosensory cortex. Thus, AC1 and AC8 have an early developmental onset with complementary spatiotemporal distribution patterns: AC1 is most broadly distributed in embryonic life, whereas AC8 is most broadly expressed in adulthood. Transient expression of these genes designate areas that may be particularly sensitive to neural activity/calcium-modulated cAMP responses during development.

Broad Spectrum Resistance to ssDNA Viruses Associated with Transgene-Induced Gene Silencing in Cassava

Geminiviruses are ssDNA viruses that infect a range of economically important crop species. We have developed a pathogen-derived transgenic approach to generate high levels of resistance against these pathogens in a susceptible cultivar of cassava (Manihot esculenta). Integration of the AC1 gene (which encodes the replication-associated protein) from African cassava mosaic virus imparted resistance against the homologous virus and provided strong cross-protection against two heterologous species of cassava-infecting geminiviruses. Short-interfering RNAs specific to the AC1 transgene were identified in the two most resistant transgenic plant lines prior to virus challenge. Levels of AC1 mRNA were suppressed in these plants. When challenged with geminiviruses, accumulation of viral DNA was reduced by up to 98% compared to controls, providing evidence that integration of AC1 initiates protection against viral infection via a post-transcriptional gene silencing mechanism. The robust cross-resistance reported has important implications for field deployment of transgenic strategies to control geminiviruses.

Gating of the cAMP signaling cascade and melatonin synthesis by the circadian clock in mammalian retina.

Melatonin is synthesized in retinal photoreceptor cells and acts as a neuromodulator imparting photoperiodic information to the retina. The synthesis of melatonin is controlled by an ocular circadian clock and by light in a finely tuned mechanism that ensures that melatonin is synthesized and acts only at night in darkness. Here we report that the circadian clock gates melatonin synthesis in part by regulating the expression of the type 1 adenylyl cyclase (AC1) and the synthesis of cAMP in photoreceptor cells. This gating is effected through E-box-mediated transcriptional activation of the AC1 gene, which undergoes robust daily fluctuations that persist in constant illumination. The circadian control of the cAMP signaling cascade indicates that the clock has a more general and profound impact on retinal functions than previously thought. In addition, rhythmic control of AC1 expression was observed in other parts of the central circadian axis, the suprachiasmatic nucleus and pineal gland, but not in other brain areas examined. Thus, clock control of the cAMP signaling cascade may play a central role in the integration of circadian signals that control physiology and behavior.

Type 8 Adenylyl Cyclase Is Targeted to Excitatory Synapses and Required for Mossy Fiber Long-Term Potentiation

Mossy fiber/CA3 long-term potentiation (LTP) is hypothesized to depend on cAMP signals generated by Ca2+-stimulated adenylyl cyclases AC1 or AC8. AC1 gene knock-out mice (AC1-/-) show a partial reduction in mossy fiber LTP, suggesting that either AC8 activity is also critical for mossy fiber LTP or that there is a component of mossy fiber LTP that is independent of CaM-activated adenylyl cyclases. To address this issue, mossy fiber LTP was examined in hippocampal slices from AC8-/- and AC1-/- x AC8-/- double knock-out mice (DKO). Despite the fact that AC8 contributes only a small fraction of the Ca2+-stimulated adenylyl cyclase activity in the hippocampus and is less sensitive to Ca2+ than AC1, AC8-/- mice exhibited mossy fiber LTP defects comparable with AC1-/- and DKO mice. Furthermore, short-term plasticity was disrupted in AC8-/- mice but not in AC1-/- mice. Because AC1 is not localized at the excitatory synapses in hippocampal neurons, we hypothesized that AC8 may be targeted to synapses, in which higher synaptic-specific Ca2+ increases occur. Here, we report that AC8 accumulates in puncta of dendrites and axons in hippocampal neurons and colocalizes with synaptic marker proteins. These data indicate that both synaptic and nonsynaptic cAMP signals, generated by different Ca2+-stimulated adenylyl cyclases, are required for mossy fiber LTP.