Physiological basis of salt stress tolerance in rice expressing the antiapoptotic gene SfIAP

Programmed cell death-associated genes, especially antiapoptosis-related genes have been reported to confer tolerance to a wide range of biotic and abiotic stresses in dicotyledonous plants such as tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.). This is the first time the antiapoptotic gene SfIAP was transformed into a monocotyledonous representative: rice (Oryza sativa L.). Transgenic rice strains expressing SfIAP were generated by the Agrobacterium-mediated transformation method and rice embryogenic calli, and assessed for their ability to confer tolerance to salt stress at both the seedling and reproductive stages using a combination of molecular, agronomical, physiological and biochemical techniques. The results show that plants expressing SfIAP have higher salt tolerance levels in comparison to the wild-type and vector controls. By preventing cell death at the onset of salt stress and maintaining the cell membrane’s integrity, SfIAP transgenic rice plants can retain plant water status, ion homeostasis, photosynthetic efficiency and growth to combat salinity successfully.

Homologs of FT, CEN and FD respond to developmental and environmental signals affecting growth and flowering in the perennial vine kiwifruit

FLOWERING LOCUS T (FT) and CENTRORADIALIS (CEN) homologs have been implicated in regulation of growth, determinacy and flowering. The roles of kiwifruit FT and CEN were explored using a combination of expression analysis, protein interactions, response to temperature in high-chill and low-chill kiwifruit cultivars and ectopic expression in Arabidopsis and Actinidia. The expression and activity of FT was opposite from that of CEN and incorporated an interaction with a FLOWERING LOCUS D (FD)-like bZIP transcription factor. Accumulation of FT transcript was associated with plant maturity and particular stages of leaf, flower and fruit development, but could be detected irrespective of the flowering process and failed to induce precocious flowering in transgenic kiwifruit. Instead, transgenic plants demonstrated reduced growth and survival rate. Accumulation of FT transcript was detected in dormant buds and stem in response to winter chilling. In contrast, FD in buds was reduced by exposure to cold. CEN transcript accumulated in developing latent buds, but declined before the onset of dormancy and delayed flowering when ectopically expressed in kiwifruit. Our results suggest roles for FT, CEN and FD in integration of developmental and environmental cues that affect dormancy, budbreak and flowering in kiwifruit.

The role of enoyl reductase genes in phloridzin biosynthesis in apple

Phloridzin is the predominant polyphenol in apple (Malus× domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

RNA interference silencing of CHS greatly alters the growth pattern of apple (Malus x domestica)

Plants produce a vast array of phenolic compounds which are essential for their survival on land. One major class of polyphenols are the flavonoids and their formation is dependent on the enzyme chalcone synthase (CHS). In a recent study we silenced the CHS genes of apple (Malus × domestica Borkh.) and observed a loss of pigmentation in the fruit skin, flowers and stems. More surprisingly, highly silenced lines were significantly reduced in size, with small leaves and shortened internode lengths. Chemical analysis also revealed that the transgenic shoots contained greatly reduced concentrations of flavonoids which are known to modulate auxin flow. An auxin transport study verified this, with an increased auxin transport in the CHS-silenced lines. Overall, these findings suggest that auxin transport in apple has adapted to take place in the presence of high endogenous concentrations of flavonoids. Removal of these compounds therefore results in abnormal auxin movement and a highly disrupted growth pattern. © 2013 Landes Bioscience.

Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes

Background Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes.

Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.

Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time

SVP-like MADS domain transcription factors have been shown to regulate flowering time and both inflorescence and flower development in annual plants, while having effects on growth cessation and terminal bud formation in perennial species. Previously, four SVP genes were described in woody perennial vine kiwifruit (Actinidia spp.), with possible distinct roles in bud dormancy and flowering. Kiwifruit SVP3 transcript was confined to vegetative tissues and acted as a repressor of flowering as it was able to rescue the Arabidopsis svp41 mutant. To characterize kiwifruit SVP3 further, ectopic expression in kiwifruit species was performed. Ectopic expression of SVP3 in A. deliciosa did not affect general plant growth or the duration of endodormancy. Ectopic expression of SVP3 in A. eriantha also resulted in plants with normal vegetative growth, bud break, and flowering time. However, significantly prolonged and abnormal flower, fruit, and seed development were observed, arising from SVP3 interactions with kiwifruit floral homeotic MADS-domain proteins. Petal pigmentation was reduced as a result of SVP3-mediated interference with transcription of the kiwifruit flower tissue-specific R2R3 MYB regulator, MYB110a, and the gene encoding the key anthocyanin biosynthetic step, F3GT1. Constitutive expression of SVP3 had a similar impact on reproductive development in transgenic tobacco. The flowering time was not affected in day-neutral and photoperiod-responsive Nicotiana tabacum cultivars, but anthesis and seed germination were significantly delayed. The accumulation of anthocyanin in petals was reduced and the same underlying mechanism of R2R3 MYB NtAN2 transcript reduction was demonstrated.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.

Remodelling the malignant phenotype : impact of EMT

Epithelial to mesenchymal transition (EMT) is a highly conserved programme of events essential for normal development. As it underpins cell plasticity, migration and invasion in a number of settings it is an attractive concept to apply to the malignant phenotype. Much is known about the molecular events regulating EMT in a number of model organisms, including Drosophila, sea urchin, zebrafish, xenopus and the chick. It is increasingly apparent that EMT is a highly conserved process, and that discoveries made in these systems provide excellent insights into the understanding of the role of EMT in human malignancies.

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.

Mice behaviorally selected for high Pavlovian fear memory exhibit fear generalization, HPA-axis alterations and increased basal activity in cortico-limbic circuits

Biological factors underlying individual variability in fearfulness and anxiety have important implications for stress-related psychiatric illness including PTSD and major depression. Using an advanced intercross line (AIL) derived from C57BL/6 and DBA/2J mouse strains and behavioral selection over 3 generations, we established two lines exhibiting High or Low fear behavior after fear conditioning. Across the selection generations, the two lines showed clear differences in training and tests for contextual and conditioned fear. Before fear conditioning training, there were no differences between lines in baseline freezing to a novel context. However, after fear conditioning High line mice demonstrated pronounced freezing in a new context suggestive of poor context discrimination. Fear generalization was not restricted to contextual fear. High fear mice froze to a novel acoustic stimulus while freezing in the Low line did not increase over baseline. Enhanced fear learning and generalization are consistent with transgenic and pharmacological disruption of the hypothalamic-pituitary-adrenal axis (HPA-axis) (Brinks, 2009, Thompson, 2004, Kaouane, 2012). To determine whether there were differences in HPA-axis regulation between the lines, morning urine samples were collected to measure basal corticosterone. Levels of secreted corticosterone in the circadian trough were analyzed by corticosterone ELISA. High fear mice were found to have higher basal corticosterone levels than low line animals. Examination of hormonal stress response components by qPCR revealed increased expression of CRH mRNA and decreased mRNA for MR and CRHR1 in hypothalamus of high fear mice. These alterations may contribute to both the behavioral phenotype and higher basal corticosterone in High fear mice. To determine basal brain activity in vivo in High and Low fear mice we used manganese-enhanced magnetic resonance imaging (MEMRI). Analysis revealed a pattern of basal brain activity made up of amygdala, cortical and hippocampal circuits that was elevated in the High line. Ongoing studies also seek to determine the relative balance of excitatory and inhibitory tone in the amygdala and hippocampus and the neuronal structure of its neurons. While these heterogeneous lines are selected on fear memory expression, HPA-axis alterations and differences in hippocampal activity segregate with the behavioral phenotypes. These differences are detectable in a basal state strongly suggesting these are biological traits underlying the behavioral phenotype (Johnson et al, 2011).

Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.

Optimising ketocarotenoid production in potato tubers : effect of genetic background, transgene combinations and environment

Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits,as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring.

Opsin clines in butterflies suggest novel roles for insect photopigments

Opsins are ancient molecules that enable animal vision by coupling to a vitamin-derived chromophore to form lightsensitive photopigments. The primary drivers of evolutionary diversification in opsins are thought to be visual tasks related to spectral sensitivity and color vision. Typically, only a few opsin amino acid sites affect photopigment spectral sensitivity. We show that opsin genes of the North American butterfly Limenitis arthemis have diversified along a latitudinal cline, consistent with natural selection due to environmental factors. We sequenced single nucleotide(SNP) polymorphisms in the coding regions of the ultraviolet (UVRh), blue (BRh), and long-wavelength (LWRh) opsin genes from ten butterfly populations along the eastern United States and found that a majority of opsin SNPs showed significant clinal variation. Outlier detection and analysis of molecular variance indicated that many SNPs are under balancing selection and show significant population structure. This contrasts with what we found by analysing SNPs in the wingless and EF-1 alpha loci, and from neutral amplified fragment length polymorphisms, which show no evidence of significant locus-specific or genome-wide structure among populations. Using a combination of functional genetic and physiological approaches, including expression in cell culture, transgenic Drosophila, UV-visible spectroscopy, and optophysiology, we show that key BRh opsin SNPs that vary clinally have almost no effect on spectral sensitivity. Our results suggest that opsin diversification in this butterfly is more consistent with natural selection unrelated to spectral tuning. Some of the clinally varying SNPs may instead play a role in regulating opsin gene expression levels or the thermostability of the opsin protein. Lastly, we discuss the possibility that insect opsins might have important, yet-to-be elucidated, adaptive functions in mediating animal responses to abiotic factors, such as temperature or photoperiod.