The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast.

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.

Targeted plant improvement through genome editing: from laboratory to field.

KEY MESSAGE: This review illustrates how far we have come since the emergence of GE technologies and how they could be applied to obtain superior and sustainable crop production. The main challenges of today's agriculture are maintaining and raising productivity, reducing its negative impact on the environment, and adapting to climate change. Efficient plant breeding can generate elite varieties that will rapidly replace obsolete ones and address ongoing challenges in an efficient and sustainable manner. Site-specific genome editing in plants is a rapidly evolving field with tangible results. The technology is equipped with a powerful toolbox of molecular scissors to cut DNA at a pre-determined site with different efficiencies for designing an approach that best suits the objectives of each plant breeding strategy. Genome editing (GE) not only revolutionizes plant biology, but provides the means to solve challenges related to plant architecture, food security, nutrient content, adaptation to the environment, resistance to diseases and production of plant-based materials. This review illustrates how far we have come since the emergence of these technologies and how these technologies could be applied to obtain superior, safe and sustainable crop production. Synergies of genome editing with other technological platforms that are gaining significance in plants lead to an exciting new, post-genomic era for plant research and production. In previous months, we have seen what global changes might arise from one new virus, reminding us of what drastic effects such events could have on food production. This demonstrates how important science, technology, and tools are to meet the current time and the future. Plant GE can make a real difference to future sustainable food production to the benefit of both mankind and our environment.

A roadmap towards a functional paradigm for learning & memory in plants.

In plants, the acquisition, processing and storage of empirical information can result in the modification of their behavior according to the nature of the stimulus, and yet this area of research remained relatively understudied until recently. As the body of evidence supporting the inclusion of plants among the higher organisms demonstrating the adaptations to accomplish these tasks keeps increasing, the resistance by traditional botanists and agricultural scientists, who were at first cautious in allowing the application of animal models onto plant physiology and development, subsides. However, the debate retains much of its heat, a good part of it originating from the controversial use of nervous system terms to describe plant processes. By focusing on the latest findings on the cellular and molecular mechanisms underlying the well established processes of Learning and Memory, recognizing what has been accomplished and what remains to be explored, and without seeking to bootstrap neuronal characteristics where none are to be found, a roadmap guiding towards a comprehensive paradigm for Learning and Memory in plants begins to emerge. Meanwhile the applications of the new field of Plant Gnosophysiology look as promising as ever.

Non-transgenic Approach to Deliver ZFNs in Seeds for Targeted Genome Engineering.

In the post-genomic era, the efficient exploitation of the available information for plant breeding is a pressing problem. The discoveries that DNA double-stranded breaks (DSBs) are both recombinagenic and mutagenic have fuelled the development of targetable zinc-finger nucleases (ZFNs), which act as molecular scissors for the induction of controlled DSBs. These powerful tools are used by researchers to accelerate mutagenesis of the normal gene loci toward the development of useful traits in plants. Seeds contain the embryo, which is a multicellular system representing a micrography of a plant. Therefore, they can serve as a foundation for applying targeted genome engineering techniques. The following single-step method describes how to deliver and express transiently ZFNs in tomato (Solanum lycopersicum) seeds using electroporation. Unlike methods that rely on tissue culture and plant regeneration after transformation, the direct delivery of ZFNs to seeds provides a high-throughput breeding technology for safe and site-specific mutagenesis. Tomato is a leading crop in the world and biotechnological advances in this species have great impact.

Targeted gene disruption coupled with metabolic screen approach to uncover the LEAFY COTYLEDON1-LIKE4 (L1L4) function in tomato fruit metabolism.

KEY MESSAGE: Functional analysis of tomato L1L4 master transcription factor resulted in important metabolic changes affecting tomato fruit quality. Tomato fruits from mutant lines bearing targeted disruption of the heterotrimeric nuclear transcription factor Y (NF-Y) transcription factor (TF) gene LEAFY-COTYLEDON1-LIKE4 (L1L4, NF-YB6), a master regulator of biosynthesis for seed storage proteins and fatty acids, were evaluated for metabolites content and morphology. Metabolic screens using LC-MS/MS-based analysis and physico-chemical methods in different L1L4 mutants of the fourth generation allowed a comparative assessment of the effects of the TF disruption. Mutagenesis resulted in fruits phenotypically similar to wild-type with subtle shape differences in the distal end protrusion and symmetry. Conversely, mutant fruits from independent lines had significant variation in moisture content, titratable acidity and overall metabolite profiles including oxalic and citric acid, fructose, ß-carotene, total polyphenols and antioxidants. Lines 6, 7 and 9 were the richest in ß-carotene and antioxidant activity, line 4 in ascorbic acid and lines 4 and 8 in succinic acid. The reduced content of the anti-nutrient oxalic acid in several mutant fruits suggests that L1L4 gene may regulate the accumulation of this compound during fruit development. Detailed LC-MS/MS analysis of mutant seeds showed substantial differences in bioactive compounds compared to wild-type seeds. Taken together, the results suggest that the L1L4 TF is a significant regulator of metabolites both in tomato fruit and seeds providing a molecular target for crop improvement. Elucidation of the candidate genes encoding key enzymes in the affected metabolic pathways aimed to facilitate the L1L4 gene network exploration and eventually lead to systems biology approaches in tomato fruit quality.

A novel arrangement of zinc finger nuclease system for in vivo targeted genome engineering: the tomato LEC1-LIKE4 gene case.

KEY MESSAGE: A selection-free, highly efficient targeted mutagenesis approach based on a novel ZFN monomer arrangement for genome engineering in tomato reveals plant trait modifications. How to achieve precise gene targeting in plants and especially in crops remains a long-sought goal for elucidating gene function and advancing molecular breeding. To address this issue, zinc finger nuclease (ZFN)-based technology was developed for the Solanum lycopersicum seed system. A ZFN architecture design with an intronic sequence between the two DNA recognition sites was evaluated for its efficiency in targeted gene mutagenesis. Custom engineered ZFNs for the developmental regulator LEAFY-COTYLEDON1-LIKE4 (L1L4) coding for the ß subunit of nuclear factor Y, when transiently expressed in tomato seeds, cleaved the target site and stimulated imperfect repair driven by nonhomologous end-joining, thus, introducing mutations into the endogenous target site. The successful in planta application of the ZFN platform resulted in L1L4 mutations which conferred heterochronic phenotypes during development. Our results revealed that sequence changes upstream of the DNA binding domain of L1L4 can lead to phenotypic diversity including fruit organ. These results underscore the utility of engineered ZFN approach in targeted mutagenesis of tomato plant which may accelerate translational research and tomato breeding.

LEC1-LIKE paralog transcription factor: how to survive extinction and fit in NF-Y protein complex.

Transcription factor function is crucial for eukaryotic systems. The presence of transcription factor families in genomes represents a significant technical challenge for functional studies. To understand their function, we must understand how they evolved and maintained by organisms. Based on genome scale searches for homologs of LEAFY COTYLEDON-LIKE (L1L; AtNF-YB6), NF-YB transcription factor, we report the discovery and annotation of a complete repertoire of thirteen novel genes that belong to the L1L paralogous gene family of Solanum lycopersicum. Gene duplication events within the species resulted in the expansion of the L1L family. Sequence and structure-based phylogenetic analyses revealed two distinct groups of L1Ls in tomato. Natural selection appears to have contributed to the asymmetric evolution of paralogs. Our results point to key differences among SlL1L paralogs in the presence of motifs, structural features, cysteine composition and expression patterns during plant and fruit development. Furthermore, differences in the binding domains of L1L members suggest that some of them evolved new binding specificities. These results reveal dramatic functional diversification of L1L paralogs for their maintenance in tomato genome. Our comprehensive insights on tomato L1L family should provide the basis for further functional and genetic experimentation.

Retinoic acid induces autophagosome maturation through redistribution of the cation-independent mannose-6-phosphate receptor.

Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggested that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic RAR and RXR receptors. RA did not affect the levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CIMPR and transiently transfected GFP and RFP full-length CIMPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CIMPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CIMPR. Transient CIMPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA. Our results suggest that RA induces AUT acidification and maturation, a process critical in the cellular autophagic mechanism.

Autophagy: a target for retinoic acids.

Autophagy is an intracellular catabolic process that responds with great sensitivity to nutrient availability, implying that certain macro- or micro-nutrients are involved. We found that retinoic acid promotes autophagosome maturation through a pathway independent from the classic nuclear retinoid receptors. Retinoic acid redistributes the cation-independent mannose-6-phosphate receptor from the trans-Golgi region to maturing autophagosomal structures inducing their acidification. Manipulation of the autophagic activity by retinoids could have enormous health implications, since they are essential dietary components and frequently used pharmaceuticals.

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles.

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.

Aging: central role for autophagy and the lysosomal degradative system.

The lysosomal network is the major intracellular proteolytic system accounting for more than 98% of long-lived bulk protein degradation and recycling particularly in tissues such as liver and muscles. Lysosomes are the final destination of intracellular damaged structures, identified and sequestered by the processes of macroautophagy and chaperone-mediated autophagy (CMA). In the process of macroautophagy, long-lived proteins and other macromolecular aggregates and damaged intracellular organelles are first engulfed by autophagosomes. Autophagosomes themselves have limited degrading capacity and rely on fusion with lysosomes. Unlike macroautophagy, CMA does not require intermediate vesicle formation and the cytosolic proteins recognized by this pathway are directly translocated to the lysosomal membrane. Aging is a universal phenomenon characterized by progressive deterioration of cells and organs due to accumulation of macromolecular and organelle damage. The continuous removal of worn-out components and replacement with newly synthesized ones ensures cellular homeostasis and delays the aging process. Growing evidence indicate that the rate of autophagosome formation and maturation and the efficiency of autophagosome/lysosome fusion decline with age. In addition, a progressive increase in intralysosomal concentration of free radicals and the age pigment lipofuscin further diminish the efficiency of lysosomal protein degradation. Therefore, integrity of the autophagosomal-lysosomal network appears to be critical in the progression of aging. Discovery of the genes involved in the process of autophagy has provided insight into the various molecular pathways that may be involved in aging and senescence. In this review, we discuss the cellular and molecular mechanisms involved in autophagy and the role of autophagosome/lysosome network in the aging process.

Oscillatory phosphorylation of yeast Fus3 MAP kinase controls periodic gene expression and morphogenesis.

Signal-transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear whether such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here, we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating-pheromone response, undergoes sustained oscillations in its phosphorylation and activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating-gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G protein signaling Sst2 and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hr, controls morphology changes that continue for a much longer time.

MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.

The mating pathway in Saccharomyces cerevisiae has been the focus of considerable research effort, yet many quantitative aspects of its regulation still remain unknown. Using an integrated approach involving experiments in microfluidic chips and computational modelling, we studied gene expression and phenotypic changes associated with the mating response under well-defined pheromone gradients. Here we report a combination of switch-like and graded pathway responses leading to stochastic phenotype determination in a specific range of pheromone concentrations. Furthermore, we show that these responses are critically dependent on mitogen-activated protein kinase (MAPK)-mediated regulation of the activity of the pheromone-response-specific transcription factor, Ste12, as well as on the autoregulatory feedback of Ste12. In particular, both the switch-like characteristics and sensitivity of gene expression in shmooing cells to pheromone concentration were significantly diminished in cells lacking Kss1, one of the MAP kinases activated in the mating pathway. In addition, the dynamic range of gradient sensing of Kss1-deficient cells was reduced compared with wild type. We thus provide unsuspected functional significance for this kinase in regulation of the mating response.

GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs.

The conserved RCN family of proteins can bind and directly regulate calcineurin, a Ca(2+)-activated protein phosphatase involved in immunity, heart growth, muscle development, learning, and other processes. Whereas high levels of RCNs can inhibit calcineurin signaling in fungal and animal cells, RCNs can also stimulate calcineurin signaling when expressed at endogenous levels. Here we show that the stimulatory effect of yeast Rcn1 involves phosphorylation of a conserved serine residue by Mck1, a member of the GSK-3 family of protein kinases. Mutations at the GSK-3 consensus site of Rcn1 and human DSCR1/MCIP1 abolish the stimulatory effects on calcineurin signaling. RCNs may therefore oscillate between stimulatory and inhibitory forms in vivo in a manner similar to the Inhibitor-2 regulators of type 1 protein phosphatase. Computational modeling indicates a biphasic response of calcineurin to increasing RCN concentration such that protein phosphatase activity is stimulated by low concentrations of phospho-RCN and inhibited by high concentrations of phospho- or dephospho-RCN. This prediction was verified experimentally in yeast cells expressing Rcn1 or DSCR1/MCIP1 at different concentrations. Through the phosphorylation of RCNs, GSK-3 kinases can potentially contribute to a positive feedback loop involving calcineurin-dependent up-regulation of RCN expression. Such feedback may help explain the large induction of DSCR1/MCIP1 observed in brain of Down syndrome individuals.