Chitosan microparticles mitigate nitrogen deficiency in tomato plants.

Nitrogen (N) deficiency is one of the most prevalent nutrient deficiencies in plants, and has a significant impact on crop yields. In this work we aimed to develop and evaluate innovative strategies to mitigate N deficiency. We studied the effect of supplementing tomato plants grown under suboptimal N nutrition with chitosan microparticles (CS-MPs) during short- and long-term periods. We observed that the supplementation with CS-MPs prevented the reduction of aerial biomass and the elongation of lateral roots (LR) triggered by N deficiency in tomato plantlets. In addition, levels of nitrates, amino acids and chlorophyll, which decreased drastically upon N deficiency, were either partial or totally restored upon CS-MPs addition to N deficient media. Finally, we showed that CS-MPs treatments increased nitric oxide (NO) levels in root tips and caused the up-regulation of genes involved in N metabolism. Altogether, we suggest that CS-MPs enhance the growth and development of tomato plants under N deficiency through the induction of biochemical and transcriptional responses that lead to increased N metabolism. We propose treatments with CS-MPs as an efficient practice focused to mitigate the nutritional deficiencies in N impoverished soils.

The DC1 Domain Protein BINUCLEATE POLLEN is Required for POLLEN Development in Arabidopsis thaliana.

The development of the male gametophyte is a tightly regulated process that requires the precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work, we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying a defective BNP alleles failed to complete mitosis II and exhibited impaired pollen germination. By yeast two-hybrid analysis and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors, we found the NAC family transcriptional regulators Vascular Plant One-Zinc Finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP; in the absence of BNP in pollen from bnp/BNP plants, VOZ1 nuclear localization is impaired. As the voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have the role of a scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators.

A mitochondrial ADXR-ADX-P450 electron transport chain is essential for maternal gametophytic control of embryogenesis in Arabidopsis.

Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

S-Nitrosation of E3 Ubiquitin Ligase Complex Components Regulates Hormonal Signalings in Arabidopsis.

E3 ubiquitin ligases mediate the last step of the ubiquitination pathway in the ubiquitin-proteasome system (UPS). By targeting transcriptional regulators for their turnover, E3s play a crucial role in every aspect of plant biology. In plants, SKP1/CULLIN1/F-BOX PROTEIN (SCF)-type E3 ubiquitin ligases are essential for the perception and signaling of several key hormones including auxins and jasmonates (JAs). F-box proteins, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and CORONATINE INSENSITIVE 1 (COI1), bind directly transcriptional repressors AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) and JASMONATE ZIM-DOMAIN (JAZ) in auxin- and JAs-depending manner, respectively, which permits the perception of the hormones and transcriptional activation of signaling pathways. Redox modification of proteins mainly by S-nitrosation of cysteines (Cys) residues via nitric oxide (NO) has emerged as a valued regulatory mechanism in physiological processes requiring its rapid and versatile integration. Previously, we demonstrated that TIR1 and Arabidopsis thaliana SKP1 (ASK1) are targets of S-nitrosation, and these NO-dependent posttranslational modifications enhance protein-protein interactions and positively regulate SCFTIR1 complex assembly and expression of auxin response genes. In this work, we confirmed S-nitrosation of Cys140 in TIR1, which was associated in planta to auxin-dependent developmental and stress-associated responses. In addition, we provide evidence on the modulation of the SCFCOI1 complex by different S-nitrosation events. We demonstrated that S-nitrosation of ASK1 Cys118 enhanced ASK1-COI1 protein-protein interaction. Overexpression of non-nitrosable ask1 mutant protein impaired the activation of JA-responsive genes mediated by SCFCOI1 illustrating the functional relevance of this redox-mediated regulation in planta. In silico analysis positions COI1 as a promising S-nitrosation target, and demonstrated that plants treated with methyl JA (MeJA) or S-nitrosocysteine (NO-Cys, S-nitrosation agent) develop shared responses at a genome-wide level. The regulation of SCF components involved in hormonal perception by S-nitrosation may represent a key strategy to determine the precise time and site-dependent activation of each hormonal signaling pathway and highlights NO as a pivotal molecular player in these scenarios.

Roles of cytochromes P450 in plant reproductive development.

The cytochrome P450 superfamily is a large enzymatic protein family that is widely distributed along diverse kingdoms. In plants, cytochrome P450 monooxygenases (CYPs) participate in a vast array of pathways leading to the synthesis and modification of multiple metabolites with variable and important functions during different stages of plant development. This includes the biosynthesis and degradation of a great assortment of compounds implicated in a variety of physiological responses, such as signaling and defense, organ patterning and the biosynthesis of structural polymers, among others. In this review, we summarize the characteristics of the different families of plant CYPs, focusing on the most recent advances in elucidating the roles of CYPs in plant growth and development and more specifically, during plant gametogenesis, fertilization and embryogenesis.

Overexpression of Arabidopsis aspartic protease APA1 gene confers drought tolerance.

Drought is an environmental stress that severely affects plant growth and crop production. Different studies have focused on drought responses but the molecular bases that regulate these mechanisms are still unclear. We report the participation of Aspartic Protease (APA1) in drought tolerance. Overexpressing APA1 Arabidopsis plants (OE-APA1), showed a phenotype more tolerant to drought compared with WT. On the contrary, apa1 insertional lines were more sensitive to this stress compared to WT plants. Morphological and physiological differences related with the water loss were observed between leaves of OE- APA1 and WT plants. OE-APA1 leaves showed lower stomata index and stomata density as well as a smaller of the stomatic aperture compared to WT plants. qPCR analysis in OE-APA1 leaves, showed higher expression levels of genes related to ABA signaling and synthesis. Analysis of plant lines expressing APA1 promoter fused to GUS showed that APA1 is expressed in epidermal and stomata cells. In summary, this work suggests that APA1 is involved in ABA-dependent response that its overexpression confers drought tolerance in Arabidopsis.

Chitosan microparticles improve tomato seedling biomass and modulate hormonal, redox and defense pathways.

Agrobiotechnology challenges involve the generation of new sustainable bioactives with emerging properties as plant biostimulants with reduced environment impact. We analyzed the potential use of recently developed chitosan microparticles (CS-MP) as growth promoters of tomato which constitutes one of the most consumed vegetable crops worldwide. Treatments of tomato seeds with CS-MP improved germination and vigor index. In addition, CS-MP sustained application triggered an improvement in root and shoot biomass reinforcing tomato performance before transplanting. The level of reactive oxygen species (ROS), antioxidant enzyme activities and defense protein markers were modulated by CS-MP treatment in tomato plantlets. Analyses of ARR5:GUS and DR5:GUS transgenic reporter tomato lines highlighted the participation of cytokinin and auxin signaling pathways during tomato root promotion mediated by CS-MP. Our findings claim a high commercial potential of CS-MP to be incorporated as a sustainable input for tomato production.

The MED30 subunit of mediator complex is essential for early plant development and promotes flowering in Arabidopsis thaliana.

Mediator is a large multiprotein complex that is required for the transcription of most, if not all, genes transcribed by RNA Polymerase II. A core set of subunits is essential to assemble a functional Mediator in vitro and, therefore, the corresponding loss-of-function mutants are expected to be lethal. The MED30 subunit is essential in animal systems, but is absent in yeast. Here, we report that MED30 is also essential for both male gametophyte and embryo development in the model plant Arabidopsis thaliana Mutant med30 pollen grains were viable and some were able to germinate and target the ovules, although the embryos aborted shortly after fertilization, suggesting that MED30 is important for the paternal control of early embryo development. When gametophyte defects were bypassed by specific pollen complementation, loss of MED30 led to early embryo development arrest. Later in plant development, MED30 promotes flowering through multiple signaling pathways; its downregulation led to a phase change delay, downregulation of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), FLOWERING LOCUS T (FTI) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and upregulation of FLOWERING LOCUS C (FLC).

Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling.

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1-cullin-F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.

Heat stress induces ferroptosis-like cell death in plants.

In plants, regulated cell death (RCD) plays critical roles during development and is essential for plant-specific responses to abiotic and biotic stresses. Ferroptosis is an iron-dependent, oxidative, nonapoptotic form of cell death recently described in animal cells. In animal cells, this process can be triggered by depletion of glutathione (GSH) and accumulation of lipid reactive oxygen species (ROS). We investigated whether a similar process could be relevant to cell death in plants. Remarkably, heat shock (HS)-induced RCD, but not reproductive or vascular development, was found to involve a ferroptosis-like cell death process. In root cells, HS triggered an iron-dependent cell death pathway that was characterized by depletion of GSH and ascorbic acid and accumulation of cytosolic and lipid ROS. These results suggest a physiological role for this lethal pathway in response to heat stress in Arabidopsis thaliana The similarity of ferroptosis in animal cells and ferroptosis-like death in plants suggests that oxidative, iron-dependent cell death programs may be evolutionarily ancient.

The DC1-domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis.

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain-containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine- and histidine-rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre-vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 - a member of the SNARE vesicle-associated protein family and previously related to a sterol-binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants.

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

oiwa, a female gametophytic mutant impaired in a mitochondrial manganese-superoxide dismutase, reveals crucial roles for reactive oxygen species during embryo sac development and fertilization in Arabidopsis.

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.

A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia.

BACKGROUND: Tilapia (Oreochromis mossambicus) are euryhaline fishes capable of tolerating large salinity changes. In a previous study aimed to identify genes involved in osmotolerance, we isolated an mRNA sequence with similarity to GRAIL (Gene Related to Anergy In Lymphocytes), which is a critical regulator of adaptive immunity and development. Tilapia GRAIL contains a PA (protease associated) domain and a C3H2C3 RING finger domain indicative of E3 ubiquitin ligase activity. SCOPE OF REVIEW: Western blots analysis was used to assess GRAIL expression pattern and responses to hyperosmotic stress. Immunohistochemistry was used to reveal the cellular localization of GRAIL in gill epithelium. Overexpression in HEK293 T-Rex cells was used to functionally characterize tilapia GRAIL. Salinity stress causes strong up-regulation of both mRNA and protein levels of tilapia GRAIL in gill epithelium. Tissue distribution of GRAIL protein is mainly confined to gill epithelium, which is the primary tissue responsible for osmoregulation of teleost fishes. Overexpression of tilapia GRAIL in HEK293 cells increases cell survival (cell viability) while decreases apoptosis during salinity challenge. MAJOR CONCLUSIONS: Our data indicate that tilapia GRAIL is a novel E3 ubiquitin ligase involved in osmotic stress signaling, which promotes environmental salinity tolerance by supporting gill cell function during hyperosmotic stress. GENERAL SIGNIFICANCE: Involvement of tilapia GRAIL in the osmotic stress response suggests that GRAIL E3 ubiquitin ligases play a broader role in environmental stress responses, beyond their documented functions in adaptive immunity and development.

A novel tilapia prolactin receptor is functionally distinct from its paralog.

A novel tilapia prolactin (PRL) receptor (OmPRLR2) was identified based on its induction during hyperosmotic stress. OmPRLR2 protein shows 28% identity to tilapia OmPRLR1 and 26% identity to human PRLR. Comparison of OmPRLR1 and OmPRLR2 revealed conserved features of cytokine class I receptors (CKR1): a WS domain and transmembrane domain, two pairs of cysteines and N-glycosylation motifs in the extracellular region, CKR1 boxes I and II, and three tyrosines in the intracellular region. However, OmPRLR2 lacked the ubiquitin ligase and 14-3-3 binding motifs. OmPRLR2 mRNA was present in all tissues analyzed, with highest expression in gills, intestine, kidney and muscle, similar to OmPRLR1. Transfer of fish from fresh water to sea water transiently increased gill OmPRLR2 mRNA levels within 4 h but decreased its protein abundance in the long term. OmPRLR2 is expressed in part as a truncated splice variant of 35 kDa in addition to the 55 kDa full-length protein. Cloning of the mRNA encoding the 35 kDa variant revealed that it lacks the extracellular region. It is expressed at significantly higher levels in males than in females. In stably transfected HEK293 cells over-expressing tetracycline-inducible OmPRLR1 and OmPRLR2, activation of these receptors by tilapia PRL177 and PRL188 triggered different downstream signaling pathways. Moreover, OmPRLR2 significantly increased HEK293 salinity tolerance. Our data reveal that tilapia has two PRLR genes whose protein products respond uniquely to PRL and activate different downstream pathways. Expression of a short PRLR2 variant may serve to inhibit PRL binding during osmotic stress and in male tissues.

Osmotic stress sensing and signaling in fishes.

In their aqueous habitats, fish are exposed to a wide range of osmotic conditions and differ in their abilities to respond adaptively to these variations in salinity. Fish species that inhabit environments characterized by significant salinity fluctuation (intertidal zone, estuaries, salt lakes, etc.) are euryhaline and able to adapt to osmotic stress. Adaptive and acclimatory responses of fish to salinity stress are based on efficient mechanisms of osmosensing and osmotic stress signaling. Multiple osmosensors, including calcium sensing receptor likely act in concert to convey information about osmolality changes to downstream signaling and effector mechanisms. The osmosensory signal transduction network in fishes is complex and includes calcium, mitogen-activated protein kinase, 14-3-3 and macromolecular damage activated signaling pathways. This network controls, among other targets, osmosensitive transcription factors such as tonicity response element binding protein and osmotic stress transcription factor 1, which, in turn, regulate the expression of genes involved in osmotic stress acclimation. In addition to intracellular signaling mechanisms, the systemic response to osmotic stress in euryhaline fish is coordinated via hormone- and paracrine factor-mediated extracellular signaling. Overall, current insight into osmosensing and osmotic stress-induced signal transduction in fishes is limited. However, euryhaline fish species represent excellent models for answering critical emerging questions in this field and for elucidating the underlying molecular mechanisms of osmosensory signal transduction.

Functional genomics and proteomics of the cellular osmotic stress response in 'non-model' organisms.

All organisms are adapted to well-defined extracellular salinity ranges. Osmoregulatory mechanisms spanning all levels of biological organization, from molecules to behavior, are central to salinity adaptation. Functional genomics and proteomics approaches represent powerful tools for gaining insight into the molecular basis of salinity adaptation and euryhalinity in animals. In this review, we discuss our experience in applying such tools to so-called 'non-model' species, including euryhaline animals that are well-suited for studies of salinity adaptation. Suppression subtractive hybridization, RACE-PCR and mass spectrometry-driven proteomics can be used to identify genes and proteins involved in salinity adaptation or other environmental stress responses in tilapia, sharks and sponges. For protein identification in non-model species, algorithms based on sequence homology searches such as MSBLASTP2 are most powerful. Subsequent gene ontology and pathway analysis can then utilize sets of identified genes and proteins for modeling molecular mechanisms of environmental adaptation. Current limitations for proteomics in non-model species can be overcome by improving sequence coverage, N- and C-terminal sequencing and analysis of intact proteins. Dependence on information about biochemical pathways and gene ontology databases for model species represents a more severe barrier for work with non-model species. To minimize such dependence, focusing on a single biological process (rather than attempting to describe the system as a whole) is key when applying 'omics' approaches to non-model organisms.

Specific TSC22 domain transcripts are hypertonically induced and alternatively spliced to protect mouse kidney cells during osmotic stress.

We recently cloned a novel osmotic stress transcription factor 1 (OSTF1) from gills of euryhaline tilapia (Oreochromis mossambicus) and demonstrated that acute hyperosmotic stress transiently increases OSTF1 mRNA and protein abundance [Fiol DF, Kültz D (2005) Proc Natl Acad Sci USA102, 927-932]. In this study, a genome-wide search was conducted to identify nine distinct mouse transforming growth factor (TGF)-beta-stimulated clone 22 domain (TSC22D) transcripts, including glucocorticoid-induced leucine zipper (GILZ), that are orthologs of OSTF1. These nine TSC22D transcripts are encoded at four loci on chromosomes 14 (TSC22D1, two splice variants), 3 (TSC22D2, four splice variants), X (TSC22D3, two splice variants), and 5 (TSC22D4). All nine mouse TSC22D transcripts are expressed in renal cortex, medulla and papilla, and in the mIMCD3 cell line. The two TSC22D3 transcripts (including GILZ) are upregulated by aldosterone but not by hyperosmolality in mIMCD3 cells. In contrast, TSC22D4 is stably upregulated by hyperosmolality in mIMCD3 cells and increased in renal papilla compared with cortex. Moreover, all four TSC22D2 transcripts are transiently upregulated by hyperosmolality and resemble tilapia OSTF1 in this regard. All TSC22D2 transcripts depend on hypertonicity as the signal for their upregulation and are unresponsive to increases in cell-permeable osmolytes. mRNA stabilization is the mechanism for TSC22D2 upregulation by hyperosmolality. Overexpression of TSC22D2-4 in mIMCD3 cells confers protection towards osmotic stress, as evidenced by a 2.7-fold increase in cell survival after 3 days at 600 mOsmol x kg(-1). Based on variable responsiveness to aldosterone and hyperosmolality in kidney cells we conclude that mouse TSC22D genes have diverse physiological functions. TSC22D2 and TSC22D4 are involved in adaptation of renal cells to hypertonicity suggesting that they represent important elements of osmosensory signal transduction in mouse kidney cells.

Regulation of osmotic stress transcription factor 1 (Ostf1) in tilapia (Oreochromis mossambicus) gill epithelium during salinity stress.

Mechanisms of induction of osmotic stress transcription factor 1 (Ostf1) were analyzed in gill epithelium of tilapia exposed to salinity stress. Experiments with primary cultures of gill epithelial cells revealed that hyperosmotic Ostf1 induction was independent of systemic factors. In addition, the synthetic glucocorticoid receptor agonist dexamethasone did not affect Ostf1 levels, arguing against cortisol being the signal for Ostf1 induction during hyperosmotic stress. Exposure of primary gill cell cultures to a hyperosmotic agent that is cell permeable and non-hypertonic (glycerol) did not trigger Ostf1 induction. However, when gill cells were exposed to hypertonicity (either in the form of NaCl or other forms) Ostf1 was rapidly and significantly induced. Analysis of hnRNA and mRNA levels revealed that Ostf1 upregulation in gill cells of intact fish and primary cultures of gill epithelial cells was mediated by transient mRNA stabilization. In addition to the initial transient mRNA stabilization a subsequent transcriptional induction of Ostf1 was observed. In cultured gill cells increase in Ostf1 mRNA synthesis was stable and very potent, whereas in gill cells of intact fish this increase was transient. This observation suggests positive feedback by Ostf1 or one of its targets and negative feedback by systemic factors on Ostf1 transcription. We conclude that Ostf1 induction in gill epithelial cells of tilapia exposed to salinity stress (1) is independent of cortisol or other systemic factors; (2) depends on hypertonicity as the signal; and (3) is based on transient mRNA stabilization. Moreover, our data on primary cell cultures show that systemic signals are necessary to prevent sustained transcriptional induction of Ostf1 during hyperosmotic stress, indicating feedback regulation and a high degree of complexity of osmosensing and signaling networks in euryhaline fishes.

Identification and pathway analysis of immediate hyperosmotic stress responsive molecular mechanisms in tilapia (Oreochromis mossambicus) gill.

Salinity is a major environmental factor that strongly influences cellular and organismal function. We have used the euryhaline fish Oreochromis mossambicus to identify and annotate immediate hyperosmotic stress responsive molecular mechanisms and biological processes in gill epithelial cells. Using a suppression subtractive hybridization (SSH) approach, we have identified and cloned 20 novel immediate early genes whose mRNAs are induced in gill epithelial cells 4 h after transfer of fish from freshwater (FW) to seawater (SW). Full-length or partial sequences of open reading frames (ORFs) were obtained using the rapid amplification of cDNA ends (RACE) technique. Kinetics of induction was analyzed for all hyperosmotic stress-induced genes. Most genes show a robust transient increase in mRNA abundance characteristic of immediate early stress response genes with peak levels observed between 2 and 8 h after seawater transfer. The newly identified genes were classified according to their sequence similarity with other vertebrate homologs and based on their predicted functions. Pathway analysis revealed that more than half of the identified immediate hyperosmotic stress genes interact closely within a cellular stress response signaling network. Moreover, the 20 genes cluster together in six molecular processes that are rapidly activated in tilapia gills upon salinity transfer. These processes are (1) stress response signal transduction, (2) compatible organic osmolyte accumulation, (3) energy metabolism, (4) lipid transport and cell membrane protection, (5) actin-based cytoskeleton dynamics, and (6) protein and mRNA stability. Our identification and analysis of a set of novel osmo-responsive tilapia genes provides insight into critical physiological processes and pathways constituting the hyperosmotic stress adaptation program in gill epithelial cells of euryhaline fishes.