An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Functional characterization of calmodulin-like proteins, CML13 and CML14, as novel light chains of Arabidopsis class VIII myosins.

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

Chilling induces sugar and ABA accumulation that antagonistically signals for symplastic connection of dormant potato buds.

Endodormancy (ED) is a crucial stage in the life cycle of many perennial plants. ED release requires accumulating a certain amount of cold exposure, measured as chilling units. However, the mechanism governing the effect of chilling on ED duration is poorly understood. We used the potato tuber model to investigate the response to chilling as associated with ED release. We measured the accumulation of specific sugars during and after chilling, defined as sugar units. We discovered that ED duration correlated better with sugar units accumulation than chilling units. A logistic function was developed based on sugar units measurements to predict ED duration. Knockout or overexpression of the vacuolar invertase gene (StVInv) unexpectedly modified sugar units levels and extended or shortened ED, respectively. Silencing the energy sensor SNF1-related protein kinase 1, induced higher sugar units accumulation and shorter ED. Sugar units accumulation induced by chilling or transgenic lines reduced plasmodesmal (PD) closure in the dormant bud meristem. Chilling or knockout of abscisic acid (ABA) 8'-hydroxylase induced ABA accumulation, in parallel to sweetening, and antagonistically promoted PD closure. Our results suggest that chilling induce sugar units and ABA accumulation, resulting in antagonistic signals for symplastic connection of the dormant bud.

Phytocannabinoid Compositions from Cannabis Act Synergistically with PARP1 Inhibitor against Ovarian Cancer Cells In Vitro and Affect the Wnt Signaling Pathway.

Ovarian cancer (OC) is the single most lethal gynecologic malignancy. Cannabis sativa is used to treat various medical conditions, and is cytotoxic to a variety of cancer types. We sought to examine the effectiveness of different combinations of cannabis compounds against OC. Cytotoxic activity was determined by XTT assay on HTB75 and HTB161 cell lines. Apoptosis was determined by flow cytometry. Gene expression was determined by quantitative PCR and protein localization by confocal microscopy. The two most active fractions, F5 and F7, from a high Δ9-tetrahydrocannabinol (THC) cannabis strain extract, and their standard mix (SM), showed cytotoxic activity against OC cells and induced cell apoptosis. The most effective phytocannabinoid combination was THC+cannabichromene (CBC)+cannabigerol (CBG). These fractions acted in synergy with niraparib, a PARP inhibitor, and were ~50-fold more cytotoxic to OC cells than to normal keratinocytes. The F7 and/or niraparib treatments altered Wnt pathway-related gene expression, epithelial-mesenchymal transition (EMT) phenotype and ß-catenin cellular localization. The niraparib+F7 treatment was also effective on an OC patient's cells. Given the fact that combinations of cannabis compounds and niraparib act in synergy and alter the Wnt signaling pathway, these phytocannabinoids should be examined as effective OC treatments in further pre-clinical studies and clinical trials.

Cannabis-Derived Compounds Cannabichromene and Δ9-Tetrahydrocannabinol Interact and Exhibit Cytotoxic Activity against Urothelial Cell Carcinoma Correlated with Inhibition of Cell Migration and Cytoskeleton Organization.

Cannabis sativa contains more than 500 constituents, yet the anticancer properties of the vast majority of cannabis compounds remains unknown. We aimed to identify cannabis compounds and their combinations presenting cytotoxicity against bladder urothelial carcinoma (UC), the most common urinary system cancer. An XTT assay was used to determine cytotoxic activity of C. sativa extracts on T24 and HBT-9 cell lines. Extract chemical content was identified by high-performance liquid chromatography (HPLC). Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and cell cycle, using stained F-actin and nuclei. Scratch and transwell assays were used to determine cell migration and invasion, respectively. Gene expression was determined by quantitative Polymerase chain reaction (PCR). The most active decarboxylated extract fraction (F7) of high-cannabidiol (CBD) C. sativa was found to contain cannabichromene (CBC) and Δ9-tetrahydrocannabinol (THC). Synergistic interaction was demonstrated between CBC + THC whereas cannabinoid receptor (CB) type 1 and type 2 inverse agonists reduced cytotoxic activity. Treatments with CBC + THC or CBD led to cell cycle arrest and cell apoptosis. CBC + THC or CBD treatments inhibited cell migration and affected F-actin integrity. Identification of active plant ingredients (API) from cannabis that induce apoptosis and affect cell migration in UC cell lines forms a basis for pre-clinical trials for UC treatment.

Initial proplastid-to-chloroplast differentiation in the developing vegetative shoot apical meristem of Arabidopsis.

In dicot plants, the process by which undifferentiated plastids, termed proplastids, differentiate into mature functional chloroplasts begins in the shoot apical meristem (SAM) and young leaf primordia, and continues along leaf development. In this work, we followed initial chloroplast biogenesis in cells of the SAM in Arabidopsis, during the early stages of germination, using chlorophyll fluorescence as a marker. We found that cells bound to form the SAM in the mature seed embryo lack chlorophyll, while plastids in the rest of the embryo cells are somewhat developed. The initial appearance of chlorophyll in the SAM occurred two days after the onset of germination, was not expedited by higher light intensities, and required a light period of between five to ten hours within these two days. In addition, we found that biogenesis of chloroplasts occurred only in the upper layer of the SAM, as opposed to the two central subtending cell layers. This pattern was maintained, mirroring the developmental status of plastids in the mature vegetative SAM. The work also presents another model for studying proplastid-to-chloroplast development, in which differentiation can be followed in SAM cells in a defined time-wise fashion.

Adaptation of Bacillus species to dairy associated environment facilitates their biofilm forming ability.

Biofilm-forming Bacillus species are often involved in contamination of dairy products and therefore present a major microbiological challenge in the field of food quality and safety. In this study, we sequenced and analyzed the genomes of milk- and non-milk-derived Bacillus strains, and evaluated their biofilm-formation potential in milk. Unlike non-dairy Bacillus isolates, the dairy-associated Bacillus strains were characterized by formation of robust submerged and air-liquid interface biofilm (pellicle) during growth in milk. Moreover, genome comparison analysis revealed notable differences in putative biofilm-associated determinants between the dairy and non-dairy Bacillus isolates, which correlated with biofilm phenotype. These results suggest that biofilm formation by Bacillus species might represent a presumable adaptation strategy to the dairy environment.

Myosin XI-K is involved in root organogenesis, polar auxin transport, and cell division.

The interplay between myosin- and auxin-mediated processes was investigated by following root development in the triple myosin knockout mutant xi-k xi-1 xi-2 (3KO). It was found that the 3KO plants generated significantly more lateral and adventitious roots than the wild-type plants or the rescued plant line expressing functional myosin XI-K:yellow fluorescent protein (YFP; 3KOR). Using the auxin-dependent reporter DR5:venus, a significant change in the auxin gradient toward the root tip was found in 3KO plants, which correlated with the loss of polar localization of the auxin transporter PIN1 in the stele and with the increased number of stele cells with oblique cell walls. Interestingly, myosin XI-K:YFP was localized to the cell division apparatus in the root and shoot meristems. In anaphase and early telophase, XI-K:YFP was concentrated in the midzone and the forming cell plate. In late telophase, XI-K:YFP formed a ring that overlapped with the growing phragmoplast. Myosin receptors MyoB1 and MyoB2 that are highly expressed throughout the plant were undetectable in dividing cells, suggesting that the myosin function in cell division relies on distinct adaptor proteins. These results suggest that myosin XIs are involved in orchestrating root organogenesis via effects on polar distribution of auxin responses and on cell division.

Cell-cycle progress in obligate predatory bacteria is dependent upon sequential sensing of prey recognition and prey quality cues.

Predators feed on prey to acquire the nutrients necessary to sustain their survival, growth, and replication. In Bdellovibrio bacteriovorus, an obligate predator of Gram-negative bacteria, cell growth and replication are tied to a shift from a motile, free-living phase of search and attack to a sessile, intracellular phase of growth and replication during which a single prey cell is consumed. Engagement and sustenance of growth are achieved through the sensing of two unidentified prey-derived cues. We developed a novel ex vivo cultivation system for B. bacteriovorus composed of prey ghost cells that are recognized and invaded by the predator. By manipulating their content, we demonstrated that an early cue is located in the prey envelope and a late cue is found within the prey soluble fraction. These spatially and temporally separated cues elicit discrete and combinatory regulatory effects on gene transcription. Together, they delimit a poorly characterized transitory phase between the attack phase and the growth phase, during which the bdelloplast (the invaded prey cell) is constructed. This transitory phase constitutes a checkpoint in which the late cue presumably acts as a determinant of the prey's nutritional value before the predator commits. These regulatory adaptations to a unique bacterial lifestyle have not been reported previously.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

The Solanum tuberosum KST1 partial promoter as a tool for guard cell expression in multiple plant species.

To date, guard cell promoters have been examined in only a few species, primarily annual dicots. A partial segment of the potato (Solanum tuberosum) KST1 promoter (KST1 partial promoter, KST1ppro) has previously been shown to confer guard cell expression in potato, tomato (Solanum lycopersicum), citrus [Troyer citrange (C. sinensis×Poncirus trifoliata)], and Arabidopsis (Arabidopsis thaliana). Here, we describe an extensive analysis of the expression pattern of KST1ppro in eight (previously reported, as well as new) species from five different angiosperm families, including the Solanaceae and the Cucurbitaceae, Arabidopsis, the monocot barley (Hordeum vulgare), and two perennial species: grapevine (Vitis vinifera) and citrus. Using confocal imaging and three-dimensional movies, we demonstrate that KST1ppro drives guard cell expression in all of these species, making it the first dicot-originated guard cell promoter shown to be active in a monocot and the first promoter reported to confer guard cell expression in barley and cucumber (Cucumis sativus). The results presented here indicate that KST1ppro can be used to drive constitutive guard cell expression in monocots and dicots and in both annual and perennial plants. In addition, we show that the KST1ppro is active in guard cells shortly after the symmetric division of the guard mother cell and generates stable expression in mature guard cells. This allows us to follow the spatial and temporal distribution of stomata in cotyledons and true leaves.

Expression of MAX2 under SCARECROW promoter enhances the strigolactone/MAX2 dependent response of Arabidopsis roots to low-phosphate conditions.

MAIN CONCLUSION: MAX2/strigolactone signaling in the endodermis and/or quiescent center of the root is partially sufficient to exert changes in F-actin density and cellular trafficking in the root epidermis, and alter gene expression during plant response to low Pi conditions. Strigolactones (SLs) are a new group of plant hormones that regulate different developmental processes in the plant via MAX2, an F-box protein that interacts with their receptor. SLs and MAX2 are necessary for the marked increase in root-hair (RH) density in seedlings under conditions of phosphate (Pi) deprivation. This marked elevation was associated with an active reduction in actin-filament density and endosomal movement in root epidermal cells. Also, expression of MAX2 under the SCARECROW (SCR) promoter was sufficient to confer SL sensitivity in roots, suggesting that SL signaling pathways act through a root-specific, yet non-cell-autonomous regulatory mode of action. Here we show evidence for a non-cell autonomous signaling of SL/MAX2, originating from the root endodermis, and necessary for seedling response to conditions of Pi deprivation. SCR-derived expression of MAX2 in max2-1 mutant background promoted the root low Pi response, whereas supplementation of the synthetic SL GR24 to these SCR:MAX2 expressing lines further enhanced this response. Moreover, the SCR:MAX2 expression led to changes in actin density and endosome movement in epidermal cells and in TIR1 and PHO2 gene expression. These results demonstrate that MAX2 signaling in the endodermis and/or quiescent center is partially sufficient to exert changes in F-actin density and cellular trafficking in the epidermis, and alter gene expression under low Pi conditions.

nMAT4, a maturase factor required for nad1 pre-mRNA processing and maturation, is essential for holocomplex I biogenesis in Arabidopsis mitochondria.

Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its own intron-encoded maturase protein. A hallmark of maturases is that they are intron-specific, acting as cofactors that bind their intron-containing pre-RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron-encoded maturase open reading frames suggest that their splicing in vivo is assisted by 'trans'-acting protein factors. Interestingly, angiosperms harbor several nuclear-encoded maturase-related (nMat) genes that contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.

Abscission of flowers and floral organs is closely associated with alkalization of the cytosol in abscission zone cells.

In vivo changes in the cytosolic pH of abscission zone (AZ) cells were visualized using confocal microscopic detection of the fluorescent pH-sensitive and intracellularly trapped dye, 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), driven by its acetoxymethyl ester. A specific and gradual increase in the cytosolic pH of AZ cells was observed during natural abscission of flower organs in Arabidopsis thaliana and wild rocket (Diplotaxis tenuifolia), and during flower pedicel abscission induced by flower removal in tomato (Solanum lycopersicum Mill). The alkalization pattern in the first two species paralleled the acceleration or inhibition of flower organ abscission induced by ethylene or its inhibitor 1-methylcyclopropene (1-MCP), respectively. Similarly, 1-MCP pre-treatment of tomato inflorescence explants abolished the pH increase in AZ cells and pedicel abscission induced by flower removal. Examination of the pH changes in the AZ cells of Arabidopsis mutants defective in both ethylene-induced (ctr1, ein2, eto4) and ethylene-independent (ida, nev7, dab5) abscission pathways confirmed these results. The data indicate that the pH changes in the AZ cells are part of both the ethylene-sensitive and -insensitive abscission pathways, and occur concomitantly with the execution of organ abscission. pH can affect enzymatic activities and/or act as a signal for gene expression. Changes in pH during abscission could occur via regulation of transporters in AZ cells, which might affect cytosolic pH. Indeed, four genes associated with pH regulation, vacuolar H(+)-ATPase, putative high-affinity nitrate transporter, and two GTP-binding proteins, were specifically up-regulated in tomato flower AZ following abscission induction, and 1-MCP reduced or abolished the increased expression.

Arabidopsis response to low-phosphate conditions includes active changes in actin filaments and PIN2 polarization and is dependent on strigolactone signalling.

Strigolactones (SLs) are plant hormones that regulate the plant response to phosphate (Pi) growth conditions. At least part of SL-signalling execution in roots involves MAX2-dependent effects on PIN2 polar localization in the plasma membrane (PM) and actin bundling and dynamics. We examined PIN2 expression, PIN2 PM localization, endosome trafficking, and actin bundling under low-Pi conditions: a MAX2-dependent reduction in PIN2 trafficking and polarization in the PM, reduced endosome trafficking, and increased actin-filament bundling were detected in root cells. The intracellular protein trafficking that is related to PIN proteins but unassociated with AUX1 PM localization was selectively inhibited. Exogenous supplementation of the synthetic SL GR24 to a SL-deficient mutant (max4) led to depletion of PIN2 from the PM under low-Pi conditions. Accordingly, roots of mutants in MAX2, MAX4, PIN2, TIR3, and ACTIN2 showed a reduced low-Pi response compared with the wild type, which could be restored by auxin (for all mutants) or GR24 (for all mutants except max2-1). Changes in PIN2 polarity, actin bundling, and vesicle trafficking may be involved in the response to low Pi in roots, dependent on SL/MAX2 signalling.

Hexokinase mediates stomatal closure.

Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to balance the demand for CO2 for photosynthesis with the loss of water through transpiration. Increased guard-cell osmolarity leads to the opening of the stomata and decreased osmolarity causes the stomata to close. The role of sugars in the regulation of stomata is not yet clear. In this study, we examined the role of hexokinase (HXK), a sugar-phosphorylating enzyme involved in sugar-sensing, in guard cells and its effect on stomatal aperture. We show here that increased expression of HXK in guard cells accelerates stomatal closure. We further show that this closure is induced by sugar and is mediated by abscisic acid. These findings support the existence of a feedback-inhibition mechanism that is mediated by a product of photosynthesis, namely sucrose. When the rate of sucrose production exceeds the rate at which sucrose is loaded into the phloem, the surplus sucrose is carried toward the stomata by the transpiration stream and stimulates stomatal closure via HXK, thereby preventing the loss of precious water.

Strigolactone analog GR24 triggers changes in PIN2 polarity, vesicle trafficking and actin filament architecture.

Strigolactones (SLs) are plant hormones that regulate shoot and root development in a MAX2-dependent manner. The mechanism underlying SLs' effects on roots is unclear. We used root hair elongation to measure root response to SLs. We examined the effects of GR24 (a synthetic, biologically active SL analog) on localization of the auxin efflux transporter PIN2, endosomal trafficking, and F-actin architecture and dynamics in the plasma membrane (PM) of epidermal cells of the primary root elongation zone in wildtype (WT) Arabidopsis and the SL-insensitive mutant max2. We also recorded the response to GR24 of trafficking (tir3), actin (der1) and PIN2 (eir1) mutants. GR24 increased polar localization of PIN2 in the PM of epidermal cells and accumulation of PIN2-containing brefeldin A (BFA) bodies, increased ARA7-labeled endosomal trafficking, reduced F-actin bundling and enhanced actin dynamics, all in a MAX2-dependent manner. Most of the der1 and tir3 mutant lines also displayed reduced sensitivity to GR24 with respect to root hair elongation. We suggest that SLs increase PIN2 polar localization, PIN2 endocytosis, endosomal trafficking, actin debundling and actin dynamics in a MAX2-dependent fashion. This enhancement might underlie the WT root's response to SLs, and suggests noncell autonomous activity of SLs in roots.

A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds.

BACKGROUND: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Integrated transcriptome and cell phenotype analysis suggest involvement of PARP1 cleavage, Hippo/Wnt, TGF-ß and MAPK signaling pathways in ovarian cancer cells response to cannabis and PARP1 inhibitor treatment.

Introduction: Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7') and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3. Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels. Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-ß/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage. Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.

Microarray analysis revealed upregulation of nitrate reductase in juvenile cuttings of Eucalyptus grandis, which correlated with increased nitric oxide production and adventitious root formation.

The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), which is involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO was observed upon excision, which was higher in juvenile cuttings than in mature ones. Treatment with an NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production in intact plants. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to increased ability to produce NO and to form adventitious roots. Arabidopsis transgenic plants constantly expressing EgNIA did not exhibit a significantly higher lateral or adventitious root formation, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root differentiation.