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.

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.

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).

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.

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.

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.

Rapid hyperosmotic coinduction of two tilapia (Oreochromis mossambicus) transcription factors in gill cells.

Gills of euryhaline teleosts are excellent models for studying osmotic-stress adaptation because they directly contact the aquatic environment and are an important effector tissue during osmotic stress. We acclimated tilapia (Oreochromis mossambicus) from fresh water (FW) to seawater (SW); performed suppression subtractive hybridization of gill mRNAs; and identified two transcription factors, osmotic stress transcription factor 1 (OSTF1) and the tilapia homolog of transcription factor II B (TFIIB), that are rapidly and transiently induced during hyperosmotic stress. mRNA levels increase 6-fold for OSTF1 and 4-fold for TFIIB, and they reach maxima 2 h after SW transfer. Protein levels increase 7.5-fold for OSTF1 and 9-fold for TFIIB, and they reach maxima 4 h after SW transfer. Induction of OSTF1 and TFIIB increases gradually with increasing salinity. Induction of OSTF1 and TFIIB is specific for osmotic stress and absent during oxidative stress (1 mM H2O2) or heat shock (+10 degrees C). Bioinformatic analysis of OSTF1 reveals that it is a transcription factor of the TGF-beta-stimulated clone 22/GILZ family. Because some mammalian homologs are strongly induced by glucocorticoids, OSTF1 may represent the molecular link between the SW hormone cortisol and transcriptional regulation of ion transport and cell differentiation in teleost gills. Coinduction of OSTF1 and TFIIB may serve to recruit TFIIB preferentially to OSTF1 target genes during hyperosmotic stress and compensate for reduced rates of transcription resulting from salt-induced chromatin compaction. We conclude that OSTF1 and TFIIB are critical elements of osmosensory signal transduction in euryhaline teleosts that mediate osmotic adaptation by means of transcriptional regulation.

A CDPK type protein kinase is involved in rice SPS light modulation.

A protein kinase activity that can phosphorylate and inactivate rice (Oryza sativa) sucrose-phosphate synthase (SPS; UDP-glucose: d-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) was measured in extracts prepared from leaves exposed to light-dark transitions. Enzyme activity present in extracts from dark leaves was about 5-fold higher than the activity in extracts from leaves that had been collected in the light. The protein kinase (named R-SPSK) was purified about 100-fold from dark leaves and its biochemical properties were studied. The micromolar dependence of Ca2+ exhibited by R-SPSK, and its response to calmodulin antagonists was similar to the properties associated with members of the plant Calcium-Dependent Protein Kinase (CDPK) family. Two modulators of SPS activity, Pi and Glc-6-P, were examined for an effect on R-SPSK. While Glc-6-P did not affect R-SPSK activity, Pi drastically increased the kinase activity. Taken together, these data provide evidence that SPS may be regulated by a CDPK type protein-kinase whose activity is modulated by light-dark transitions and stimulated by Pi, the negative effector of SPS activity.