Application of a JA-Ile Biosynthesis Inhibitor to Methyl Jasmonate-Treated Strawberry Fruit Induces Upregulation of Specific MBW Complex-Related Genes and Accumulation of Proanthocyanidins.

Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA (jasmonoyl-isoleucine, JA-Ile) biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry (Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related (FabHLH3/33, FaMYB9/10/11, and repressor FaMYB1), and anthocyanin and PA biosynthetic (FaANS, FaUFGT, FaANR, and FaLAR) genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.

Bacterial Production of a Pederin Analogue by a Free-Living Marine Alphaproteobacterium.

The polyketide pederin family are cytotoxic compounds isolated from insects, lichen, and marine sponges. During the past decade, different uncultivable bacteria symbionts have been proposed as the real producers of these compounds, such as those found in insects, lichen, and marine sponges, and their trans-AT polyketide synthase gene clusters have been identified. Herein we report the isolation and biological activities of a new analogue of the pederin family, compound 1, from the culture of a marine heterotrophic alphaproteobacterium, Labrenzia sp. PHM005. This is the first report of the production of a pederin-type compound by a free-living marine bacteria that could be cultured in the laboratory.

Abscisic Acid Synthesis and Signaling during the Ripening of Raspberry (Rubus idaeus 'Heritage') Fruit.

The raspberry (Rubus idaeus L.) fruit is characterized by its richness in functional molecules and high nutritional value, but the high rate of fruit softening limits its quality during postharvest. Raspberry drupelets have a particular ripening regulation, depending partially on the effect of ethylene produced from the receptacle. However, the possible role of abscisic acid (ABA) in the modulation of quality parameters during the ripening of raspberry is unclear. This study characterized the fruit quality-associated parameters and hormonal contents during fruit development in two seasons. The quality parameters showed typical changes during ripening: a drastic loss of firmness, increase in soluble solids content, loss of acidity, and turning to a red color from the large green stage to fully ripe fruit in both seasons. A significant increase in the ABA content was observed during the ripening of drupelets and receptacles, with the higher content in the receptacle of ripe and overripe stages compared to the large green stage. Moreover, identification of ABA biosynthesis-(9-cis-epoxycarotenoid dioxygenase/NCED) and ABA receptor-related genes (PYRs-like receptors) showed three genes encoding RiNCEDs and nine genes for RiPYLs. The expression level of these genes increased from the large green stage to the full-ripe stage, specifically characterized by a higher expression of RiNCED1 in the receptacle tissue. This study reports a consistent concomitant increase in the ABA content and the expression of RiNCED1, RiPYL1, and RiPYL8 during the ripening of the raspberry fruit, thus supporting the role for ABA signaling in drupelets.

Streptomyces pharmamarensis sp. nov. isolated from a marine sediment.

A Gram-stain-positive actinobacterium, strain PM267(T), was isolated from a marine sediment sample in the Mediterranean Sea. The novel strain produced extensively branched substrate and aerial hyphae that carried spiral spore chains. Substrate and aerial mycelia were cream-white and white, respectively. Diffusible pigments were not observed. 16S rRNA gene sequence analysis revealed that strain PM267(T) belonged to the genus Streptomyces and shared a gene sequence similarity of 97.1 % with Streptomyces artemisiae YIM 63135(T) and Streptomyces armeniacus JCM 3070(T). Values <97 % were obtained with other sequences representing members of the genus Streptomyces. The cell wall peptidoglycan contained ll-diaminopimelic acid. MK-9(H(8)) was the major menaquinone. The phospholipid pattern included phosphatidylethanolamine as diagnostic lipid (type II). Major fatty acids found were iso- and anteiso- fatty acids. The G+C content of the DNA was 71.2 mol%. The strain was halotolerant and was able to grow in the presence of 9 % (w/v) NaCl (with an optimum of 2 %). On the basis of these results and additional physiological data obtained in the present study, strain PM267(T) represents a novel species within the genus Streptomyces for which the name Streptomyces pharmamarensis sp. nov. is proposed (type strain PM267(T)  = CECT 7841(T)  = DSM 42032(T)).

Antitumor actinopyranones produced by Streptomyces albus POR-04-15-053 isolated from a marine sediment.

Four new antitumor pyranones, PM050511 (1), PM050463 (2), PM060054 (3), and PM060431 (4), were isolated from the cell extract of the marine-derived Streptomyces albus POR-04-15-053. Their structures were elucidated by a combination of spectroscopic methods, mainly 1D and 2D NMR and HRESIMS. They consist of an α-methoxy-γ-pyrone ring containing a highly substituted tetraene side chain glycosylated at C-10 in the case of 1 and 4. Compounds 1 and 4 displayed strong cytotoxicity against three human tumor cell lines with GI50 values in the submicromolar range, whereas 2 showed subnanomolar activity as an inhibitor of EGFR-MAPK-AP1-mediated mitogenic signaling, causing inhibition of EGF-mediated AP1 trans-activation and EGF-mediated ERK activation and slight inhibition of EGF-mediated JNK activation. Taken together, these results suggest that members of the pyranone family of compounds could be developed as potential antitumor agents.

Detection of a pederin-like compound using a dilution-to-extinction-based platform for the isolation of marine bacteria in drug discovery strategies.

The continued development of culturing technologies for the discovery of new molecules from marine microbes is of paramount importance for drug discovery. Coupled with this, the use of the high-throughput approach shows promise for increasing the number of Gram-negative and non-filamentous bacteria cultures that can be surveyed, since they show a lower potential of bioactivity. In this work, we propose a new strategy of high-throughput cultivation of bacteria inspired by a dilution-to-extinction (DTE) methodology for the isolation of, and screening for, new cytotoxic compound producing marine bacteria. A marine sponge tissue was directly used as inoculum and the results were compared with the data obtained through the direct plating isolation method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) genomic fingerprinting indicated the isolation of four bioactive strains, three of them producers of a pederin-like compound, and the fourth one able to synthesize a different compound, still unidentified, rendered by the DTE approach, in comparison with one bioactive strain identified through the plating method. Analyses based on the 16S rRNA gene data showed the existence of two different species belonging to the genus Labrenzia. The efficiency and diversity ratio in the number of isolates and compounds are discussed. In view of the results, the proposed DTE approach proved to be efficient for the isolation of new cytotoxic compounds of marine origin and pave the way for future potential applications.

Priming of Defense Systems and Upregulation of MYC2 and JAZ1 Genes after Botrytis cinerea Inoculation in Methyl Jasmonate-Treated Strawberry Fruits.

Several attempts have been made to study the effects of methyl jasmonate (MeJA) on plants in the past years. However, the comparative effects of the number and phenological time of MeJA applications on the activation of defense systems is currently unknown in strawberries. In the present research, we performed three field treatments during strawberry (Fragaria× ananassa 'Camarosa') fruit development and ripening which consisted of differential MeJA applications at flowering (M3), and the large green (M2 and M3) and red ripe (M1, M2, and M3) fruit stages. We also checked changes in gene expression related to plant defense against Botrytis cinerea inoculation post-harvest. In M3 treatment, we observed an upregulation of the anthocyanin and lignin contents and the defense-related genes, encoding for chitinases, ß-1,3-glucanases and polygalacturonase-inhibiting proteins, after harvest (0 hpi), along with the jasmonate signaling-related genes FaMYC2 and FaJAZ1 at 48 h after B. cinerea inoculation (48 hpi) during postharvest storage. Although we did not find differences in gray mold incidence between the MeJA treatments and control, these results suggest that preharvest MeJA treatment from the flowering stage onwards (M3) primes defense responses mediated by the upregulation of different defense-related genes and retains the upregulation of MYC2 and JAZ1 at 48 hpi.

Methyl Jasmonate Applications From Flowering to Ripe Fruit Stages of Strawberry (Fragaria × ananassa 'Camarosa') Reinforce the Fruit Antioxidant Response at Post-harvest.

Preharvest applications of methyl jasmonate (MeJA) have been shown to improve post-harvest fruit quality in strawberry fruit. However, the effectiveness of consecutive field applications at different phenological stages on the reinforcement of the antioxidant capacity remains to be analyzed. To determine the best antioxidant response of strawberry (Fragaria × ananassa 'Camarosa') fruit to different numbers and timing of MeJA applications, we performed three differential preharvest treatments (M1, M2, and M3) consisted of successive field applications of 250 µmol L-1 MeJA at flowering (M3), large green (M2 and M3), and ripe fruit stages (M1, M2, and M3). Then, we analyzed their effects on fruit quality parameters [firmness, skin color, soluble solids content/titratable acidity (SSC/TA) ratio, fruit weight at harvest, and weight loss] along with anthocyanin and proanthocyanidin (PA) accumulation; the antioxidant-related enzymatic activity of catalase (CAT), guaiacol peroxidase (POX), and ascorbate peroxidase (APX); the total flavonoid and phenolic contents, antioxidant capacity, and ascorbic acid content (AAC) during post-harvest storage (0, 24, 48, and 72 h). We also evaluated the effect on lignin, total carbon and nitrogen (%C and N), lipid peroxidation, and C and N isotopes signatures on fruits. Remarkably, the results indicated that MeJA treatment increases anthocyanin and PA contents as well as CAT activity in post-harvest storage, depending on the number of preharvest MeJA applications. Also, M3 fruit showed a higher AAC compared to control at 48 and 72 h. Noticeably, the anthocyanin content and CAT activity were more elevated in M3 treatment comparing with control at all post-harvest times. In turn, APX activity was found higher on all MeJA-treated fruit independent of the number of applications. Unlike, MeJA applications did not generate variations on fruit firmness and weight, lignin contents,% C and N, and in lipid peroxidation and water/nitrogen use efficiency according to C and N isotope discrimination. Finally, we concluded that an increasing number of MeJA applications (M3 treatment) improve anthocyanin, PA, AAC, and CAT activity that could play an essential role against reactive oxygen species, which cause stress that affects fruits during post-harvest storage.

Tartrolon D, a cytotoxic macrodiolide from the marine-derived actinomycete Streptomyces sp. MDG-04-17-069.

Exploration of marine-derived actinomycetes as a source of antitumor compounds has led to the isolation of a new member of the tartrolon series, tartrolon D (4). This new compound was obtained from Streptomyces sp. MDG-04-17-069 fermentation broths and displayed strong cytotoxic activity against three human tumor cell lines. Additionally, the known compound ikarugamycin (5) was also found in the culture broths of the same microorganism. The structure of this new tartrolon was established by a combination of spectroscopic techniques (1D and 2D NMR, HRMS, and UV) as well as by comparison with published data for similar compounds.

New Fluvirucinins C1 and C2 Produced by a Marine Derived Actinomycete.

Two new fluvirucin aglycones, named fluvirucinins C, and C2 (1-2), have been isolated from the ethyl acetate mycelial cake extract of the fermentation broth of.a marine sponge-associated actinomycete. Fluvirucinins C, (1) and C2 (2) represent a new type of 14-membered macrolactam aglycon, structurally related with the common aglycon of the known fluvirucins. Their structures were elucidated on the basis of ID and 2D NMR analyses, as well as HRESIMS experiments. The antimicrobial and cytotoxic activities of compounds 1 and 2 have been evaluated, but no significant activities found for fluvirucinins C, and C2.

PM100117 and PM100118, new antitumor macrolides produced by a marine Streptomyces caniferus GUA-06-05-006A.

Two new bioactive polyhydroxyl macrolide lactones PM100117 (1) and PM100118 (2) were isolated from the culture broth of the marine-derived Streptomyces caniferus GUA-06-05-006A. Their structures were elucidated by a combination of spectroscopic methods, mainly one-dimensional and 2D NMR and HRESI-MS. They consist of 36-membered macrolides with a side chain containing three deoxy sugars and a 1,4-naphthoquinone chromophore. Compounds 1 and 2 displayed potent cytotoxicity against three human tumor cell lines with GI50 values in the micromolar range, as well as slight antifungal activity against Candida albicans ATCC10231. In addition, both compounds alter the plasma membrane of tumor cells, inducing loss of membrane integrity and subsequent cell permeabilization leading to a fast and dramatic necrotic cell death.