Tamar Lotan

Pathogenesis-related proteins are developmentally regulated in tobacco flowers.

The accumulation of pathogenesis-related proteins (PR) in tobacco leaves has been casually related to pathogen and specific physiological stresses. The known enzymatic function of some of these proteins is potentially antimicrobial. By using antibodies specific to three classes of pathogenesis-related proteins, we examined tobacco plants during their normal growth. The pathogenesis-related proteins accumulated during the normal development of the tobacco flower. The PR-1 class of proteins (biological function unknown) is located in sepal tissue. PR-P, Q polypeptides are endochitinases and are present in pedicels, sepals, anthers, and ovaries. A glycoprotein serologically related to the PR-2,N,O class is a (1,3)-beta-glucanase and is present in pistils. Differential appearance during flower development, in situ localization, and post-translational processing of floral pathogenesis-related proteins point to a hitherto unsuspected function these classes of pathogenesis-related proteins play in the normal process of flowering and reproductive physiology.

Cloning and expression inEscherichia coli of the gene encoding β-C-4-oxygenase, that converts β-carotene to the ketocarotenoid canthaxanthin inHaematococcus pluvialis

In the green algaHaematococcus pluvialis the ketocarotenoid astaxanthin accumulates under stress conditions. Astaxanthin is a red carotenoid pigment which possess antioxidative activity. We have cloned the gene for β‐C‐4 oxygenase (β‐carotene ketolase) from the green algaeH. pluvialis. The cloning method took advantage of a strain ofE. coli which was genetically engineered to produce β‐carotene. An expression cDNA library ofH. pluvialis was transfected to cells of this strain and visually screened for brown‐red pigmented colonies. One colony out of 100,000 transformants showed color change due to accumulation of canthaxanthin. The cDNA clone in this transformant colony encodes the enzyme β‐C‐4 oxygenase that catalyzes the conversion of β carotene to canthaxanthin via echinenone. This enzyme does not convert zeaxanthin to astaxanthin. It is concluded that inH. pluvialis astaxanthin is synthesized via canthaxanthin and therefore an additional enzyme is predicted, which converts canthaxanthin to astaxanthin.

A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass

A novel stylar‐specific glycosylated protein, sp41, was characterized. Sp41 constitutes greater than 12% of the transmitting tract tissue soluble proteins and is mainly localized in the extracellular matrix. Two cDNA clones corresponding to sp41 mRNA were isolated and sequenced. The decoded sequences are, respectively, 80% and 49% homologous to acidic and basic pathogen‐induced (1‐3)‐beta‐glucanases of the leaf. Thus a subfamily of (1‐3)‐beta‐glucanase pathogenesis‐related (PR) proteins constitutes one of the major stylar matrix proteins. The accumulation of sp41 transcripts in normally developing and elicitor‐treated styles and leaves was followed using an RNase protection assay. During development sp41 transcript accumulation starts well after carpel differentiation. It is first detected in styles at 8 days before anthesis. The maximal level of accumulation is reached during anthesis. Elicitor‐treated styles do not accumulate the leaf‐type (1‐3)‐beta‐glucanase transcript, although they retain the capacity to synthesize leaf‐type pathogenesis‐related proteins such as the pathogen‐induced acidic chitinase. The developmental regulation of sp41 expression points to a role for them in the normal processes of flowering and reproductive physiology.

Molecular genetics of the carotenoid biosynthesis pathway in plants and algae

During recent years genes for more than 20 different carotenogenic enzymes have been cloned from various organisms: bacteria, cyanobacteria, fungi, algae and plants. This accomplishment has provided new molecular tools to study the enzymes and yielded new information on their structure, function and regulation. We describe here the recent progress in the molecular genetics of the carotenoid biosynthesis pathway in plants. To date, the genes for almost all the enzymes, from the early steps of the isoprenoid pathway to the predominant xanthophylls, have been cloned. Their characterization had an immense impact on our understanding of carotenoid biosynthesis at the molecular level.

Transcriptome profiling of the dynamic life cycle of the scypohozoan jellyfish Aurelia aurita

BackgroundThe moon jellyfish Aurelia aurita is a widespread scyphozoan species that forms large seasonal blooms. Here we provide the first comprehensive view of the entire complex life of the Aurelia Red Sea strain by employing transcriptomic profiling of each stage from planula to mature medusa.ResultsA de novo transcriptome was assembled from Illumina RNA-Seq data generated from six stages throughout the Aurelia life cycle. Transcript expression profiling yielded clusters of annotated transcripts with functions related to each specific life-cycle stage. Free-swimming planulae were found highly enriched for functions related to cilia and microtubules, and the drastic morphogenetic process undergone by the planula while establishing the future body of the polyp may be mediated by specifically expressed Wnt ligands. Specific transcripts related to sensory functions were found in the strobila and the ephyra, whereas extracellular matrix functions were enriched in the medusa due to high expression of transcripts such as collagen, fibrillin and laminin, presumably involved in mesoglea development. The CL390-like gene, suggested to act as a strobilation hormone, was also highly expressed in the advanced strobila of the Red Sea species, and in the medusa stage we identified betaine-homocysteine methyltransferase, an enzyme that may play an important part in maintaining equilibrium of the medusa’s bell. Finally, we identified the transcription factors participating in the Aurelia life-cycle and found that 70% of these 487 identified transcription factors were expressed in a developmental-stage-specific manner.ConclusionsThis study provides the first scyphozoan transcriptome covering the entire developmental trajectory of the life cycle of Aurelia. It highlights the importance of numerous stage-specific transcription factors in driving morphological and functional changes throughout this complex metamorphosis, and is expected to be a valuable resource to the community.

Early and late response of Nematostella vectensis transcriptome to heavy metals

Environmental contamination from heavy metals poses a global concern for the marine environment, as heavy metals are passed up the food chain and persist in the environment long after the pollution source is contained. Cnidarians play an important role in shaping marine ecosystems, but environmental pollution profoundly affects their vitality. Among the cnidarians, the sea anemone Nematostella vectensis is an advantageous model for addressing questions in molecular ecology and toxicology as it tolerates extreme environments and its genome has been published. Here, we employed a transcriptome‐wide RNA‐Seq approach to analyse N. vectensis molecular defence mechanisms against four heavy metals: Hg, Cu, Cd and Zn. Altogether, more than 4800 transcripts showed significant changes in gene expression. Hg had the greatest impact on up‐regulating transcripts, followed by Cu, Zn and Cd. We identified, for the first time in Cnidaria, co‐up‐regulation of immediate‐early transcription factors such as Egr1, AP1 and NF‐κB. Time‐course analysis of these genes revealed their early expression as rapidly as one hour after exposure to heavy metals, suggesting that they may complement or substitute for the roles of the metal‐mediating Mtf1 transcription factor. We further characterized the regulation of a large array of stress‐response gene families, including Hsp, ABC, CYP members and phytochelatin synthase, that may regulate synthesis of the metal‐binding phytochelatins instead of the metallothioneins that are absent from Cnidaria genome. This study provides mechanistic insight into heavy metal toxicity in N. vectensis and sheds light on ancestral stress adaptations.

Transdermal Delivery of Scopolamine by Natural Submicron Injectors: In-Vivo Study in Pig

Transdermal drug delivery has made a notable contribution to medical practice, but has yet to fully achieve its potential as an alternative to oral delivery and hypodermic injections. While transdermal delivery systems would appear to provide an attractive solution for local and systemic drug delivery, only a limited number of drugs can be delivered through the outer layer of the skin. The most difficult to deliver in this way are hydrophilic drugs. The aquatic phylum Cnidaria, which includes sea anemones, corals, jellyfish and hydra, is one of the most ancient multicellular phyla that possess stinging cells containing organelles (cnidocysts), comprising a sophisticated injection system. The apparatus is folded within collagenous microcapsules and upon activation injects a thin tubule that immediately penetrates the prey and delivers its contents. Here we show that this natural microscopic injection system can be adapted for systemic transdermal drug delivery once it is isolated from the cells and uploaded with the drug. Using a topically applied gel containing isolated natural sea anemone injectors and the muscarinic receptor antagonist scopolamine, we found that the formulated injectors could penetrate porcine skin and immediately deliver this hydrophilic drug. An in-vivo study in pigs demonstrated, for the first time, rapid systemic delivery of scopolamine, with Tmax of 30 minutes and Cmax 5 times higher than in controls treated topically with a scopolamine-containing gel without cnidocysts. The ability of the formulated natural injection system to penetrate a barrier as thick as the skin and systemically deliver an exogenous compound presents an intriguing and attractive alternative for hydrophilic transdermal drug delivery.

Immediate topical drug delivery by natural submicron injectors

Transdermal delivery is an attractive but challenging solution for delivery of drugs. The sea anemone possesses a sophisticated injection system, which utilizes built-in high osmotic pressures. The system is folded within microcapsules and upon activation it injects a long, needle-like tubule of submicron diameter that penetrates the target in a fraction of a second. Here we show that this natural injection system can be adapted for active topical drug delivery once it is isolated from the cells, formulated into a topical gel, and uploaded with the desired drug. The formulated injectors retained their physical characteristics and were capable of penetrating the skin, achieving immediate delivery of a hydrophilic compound. We demonstrate quantitative rapid delivery of lidocaine hydrochloride as a function of microcapsular and drug concentrations. The adaptation of natural injectors for drug delivery combines the benefits of short topical application with rapid delivery of physical devices, thereby presenting a promising alternative for transdermal drug delivery.

Pathogenesis-Related Proteins Exhibit Both Pathogen-Induced and Developmental Regulation

Antisera to acidic isoforms of pathogenesis-related (PR) proteins were used to measure the activity of these genes in tobacco plants. A novel endo-(1-4)-β-xylanase purified from fungal filtrates of Trichoderma viride was found to be a strong activator of PR proteins synthesis in tobacco leaves. The induction was not inhibited by blockers of either ethylene biosynthesis or ethylene action highlighting a novel ethylene independent pathway for PR proteins. Concomitant with the induction of PR proteins phytoalexins are induced. The regulation of the phytoalexin capsidiol showed identical ethylene dependent and independent pathways described for PR proteins.

Lineage-specific evolution of cnidarian Wnt ligands

We have studied the evolution of Wnt genes in cnidarians and the expression pattern of all Wnt ligands in the hydrozoan Hydractinia echinata. Current views favor a scenario in which 12 Wnt sub‐families were jointly inherited by cnidarians and bilaterians from their last common ancestor. Our phylogenetic analyses clustered all medusozoan genes in distinct, well‐supported clades, but many orthologous relationships between medusozoan Wnts and anthozoan and bilaterian Wnt genes were poorly supported. Only seven anthozoan genes, Wnt2, Wnt4, Wnt5, Wnt6, Wnt 10, Wnt11, and Wnt16 were recovered with strong support with bilaterian genes and of those, only the Wnt2, Wnt5, Wnt11, and Wnt16 clades also included medusozoan genes. Although medusozoan Wnt8 genes clustered with anthozoan and bilaterian genes, this was not well supported. In situ hybridization studies revealed poor conservation of expression patterns of putative Wnt orthologs within Cnidaria. In polyps, only Wnt1, Wnt3, and Wnt7 were expressed at the same position in the studied cnidarian models Hydra, Hydractinia, and Nematostella. Different expression patterns are consistent with divergent functions. Our data do not fully support previous assertions regarding Wnt gene homology, and suggest a more complex history of Wnt family genes than previously suggested. This includes high rates of sequence divergence and lineage‐specific duplications of Wnt genes within medusozoans, followed by functional divergence over evolutionary time scales.