The role of proteomics in progressing insights into plant secondary metabolism

The development of omics has enabled the genome-wide exploration of all kinds of biological processes at the molecular level. Almost every field of plant biology has been analyzed at the genomic, transcriptomic and proteomic level. Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago. We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies. These issues are: (i) the selection of a suitable plant material rich in secondary metabolites of interest, such as specialized tissues and organs, and in vitro cell cultures; (ii) the proteomic strategy to access target proteins, either a comprehensive or a differential analysis; (iii) the proteomic approach, represented by the hypothesis-free discovery proteomics and the hypothesis-driven targeted proteomics. We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-02; BIO2014-51861-R), European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). JM-C acknowledges a postdoctoral and research grants from SENESCYT GOVERNMENT OF ECUADOR (006-IECE-SMG5-GPLR-2012 and Programa1_Senescyt_2014) and a grant from UTEQ (UTEQ-Ambiental-9-FCAmb-IFOR-2014-FOCICYT002)

Factors Affecting the Bioproduction of Resveratrol by Grapevine Cell Cultures under Elicitation

Here we present a study of the characterization and optimization of the production of trans-Resveratrol (t-R) in grape (Vitis vinifera cv. Gamay) cell cultures elicited with methyl jasmonate (MeJA) and dimethyl-β-cyclodextrin (DIMEB). The aim of this study was to determine the influence of a number of factors of the grapevine cell culture on t-R production level in 250 mL shaken flasks that would enable the better control of this bioproduction system when it is upscaled to a 2 L stirred bioreactor. The factors included the optimal growth phase for elicitation, the concentration of elicitors and of biomass, the order of addition of elicitors, and the illumination regime and ageing of cells. We found out that the optimal biomass density for the production of t-R was 19% (w/v) with an optimal ratio of 0.5 g DIMEB/g biomass. The most productive concentrations of the elicitors tested were 50 mM DIMEB and 100 µM MeJA, reaching maximum values of 4.18 mg·mL−1 and 16.3 mg·g biomass−1 of t-R concentration and specific production, respectively. We found that the order of elicitor addition matters since, as compared with the simultaneous addition of both elicitors, the addition of MeJA 48 h before DIMEB results in ca. 40% less t-R production, whilst there is no significant difference when MeJA is added 48 h after DIMEB. Upon upscaling, the better conditions tested for t-R production were aeration at 1.7 vol/vol/min without agitation, 24 °C, and 30 g·L−1 sucrose, achieving production rates similar to those obtained in shaken flasks.This work has been supported by Spanish Ministry of Science and Innovation, MCIN/AEI/10.13039/501100011033, ERDF A way of making Europe and European Union NextGenerationEU/PRTR grants PID2020-113438RB-I00 and TED2021-129617B-I00, Valencian Conselleria d’Innovació, Universitats, Ciencia y Societat Digital grant CIAICO/2021/167

A DIGE proteomic analysis of wheat flag leaf treated with TERRA-SORB® foliar, a free amino acid high content biostimulant

The flag leaf is the most important source of carbohydrate during wheat kernel filling. Around a 75% of all sugars stored in the kernel come from carbon fixed by this leaf. Terra-Sorb® foliar is an L-α-amino acid-based product from enzymatic hydrolysis for foliar application with a high ratio of free to total amino acids. Previous agronomical studies carried out on grassy, horticultural and tree crops have shown that the application of Terra-Sorb® increases photosynthetic plant activity and chlorophyll content, promotes rapid recovery from stress and improves fruit set. In this work, we have undertaken a proteomic approach to explore molecular mechanisms potentially involved in the stimulating effect of Terra-Sorb® Foliar on wheat yield when applied in commercial fields. Wheat plants at the flag leaf stage were treated, and a DIGE approach was used to compare the proteomes of treated vs. control plants in four biological replicates. Thirty-seven protein spots were found to change in abundance (ANOVA p<0.05) out of which 8 were down-regulated and 29 up-regulated in treated leaves. Twenty protein spots (1.2<fold change <1.9) encoded by 11 different genes were successfully identified by nLC-ESI-MS/MS and NCBInr database search. The deregulated proteins identified were mainly related to the life cycle of Rubisco. Importantly, two proteins involved in the positive regulation of Rubisco activity, namely Rubisco activase, and the large subunit of Rubisco binding protein, were found up-regulated in treated plants, suggesting a better performance of Rubisco. Down-regulated proteins were of metabolic and anti-stress enzymes, including Cu/Zn superoxide dismutase that protects photosystem 11 from photooxidation. In conclusion, significant changes were shown to occur in the wheat flag leaf proteome upon Terra-Sorb® Foliar application. The deregulated proteins identified are directly or indirectly involved in the C02 fixation which may correlate with the known stimulating effect of Terra-Sorb® Foliar of wheat yield, although further functional experiments are needed to validate the proposed hypothesis.This work was supported by Bioibérica, S.A. MJME acknowledged a grant contract from Fundación CajaMurcia-Universidad de Alicante

Dispensacion de anticonceptivos hormonales orales

El 80% de las mujeres españolas en edad fértil utilizan métodos anticonceptivos. Los anticonceptivos orales hormonales son los que obtienen mayor grado de satisfacción entre las usuarias siendo el segundo método utilizado. Método Estudio observacional descriptivo en 7 farmacias de la Comunidad de Madrid. Durante seis meses (enero-junio de 2009) se realizó una entrevista en el mostrador a las usuarias de métodos anticonceptivos hormonales orales para conocer qué tipo de paciente los demanda, si existen factores de riesgo que desaconsejen su utilización y el cumplimiento de la legislación vigente en cuanto a dispensación. Los datos se registraron y se evaluaron posteriormente. Resultados Existe un alto número de usuarias (39,3%) que creen que estos medicamentos pueden ser adquiridos sin receta médica en las farmacias comunitarias, por lo que habrá que realizar más educación sanitaria al respecto

Prenylated Flavonoids of the Moraceae Family: A Comprehensive Review of Their Biological Activities

Prenylated flavonoids (PFs) are natural flavonoids with a prenylated side chain attached to the flavonoid skeleton. They have great potential for biological activities such as anti-diabetic,anti-cancer, antimicrobial, antioxidant, anti-inflammatory, enzyme inhibition, and anti-Alzheimer’s effects. Medicinal chemists have recently paid increasing attention to PFs, which have become vital for developing new therapeutic agents. PFs have quickly developed through isolation and semi- or full synthesis, proving their high value in medicinal chemistry research. This review comprehensively summarizes the research progress of PFs, including natural PFs from the Moraceae family and their pharmacological activities. This information provides a basis for the selective design and optimization of multifunctional PF derivatives to treat multifactorial diseases.This work has been supported by the Spanish Ministry of Science and Innovation, MCIN/AEI/10.13039/501100011033, ERDF A way of making Europe, and European Union NextGenerationEU/PRTR grants PID2020-113438RB-I00 and TED2021-129617B-I00, and Valencian Conselleria d’Innovació, Universitats, Ciencia y Societat Digital grant CIAICO/2021/167. This work was also supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Contract No. 451-03-66/2024-03/200007)

Optimization of Glycerol Concentration and Freezing Rate in the Cryopreservation of Ejaculate From Brown Bear (Ursus arctos)

P. 105-112In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES–Tris–Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at −10, −20 or −40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre‐freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of −20°C/min for freezing brown bear ejaculated spermatozoa.S

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide, and Importance of Individual Male Variability

P. 393-403Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H2O2) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H2O2 for 2 h at 37°C. Intracellular reactive oxygen species (H2DCFDA‐CM) increased with H2O2 concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H2O2 and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H2O2 for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H2O2 presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H2O2 or after incubation with H2O2. Red deer spermatozoa are relatively resilient to H2O2 after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.S

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t-resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β-cyclodextrins

The growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.This work has been supported by a grant from the Spanish Ministry of Science and Innovation (BIO2014-51861-R). Diego Hidalgo is a predoctoral fellow of Mexican CONACYT

DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification

Introduction and objectiveCryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species.MethodsTestes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope.Results and discussionOur preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls.ConclusionsIn conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type

Berry Flesh and Skin Ripening Features in Vitis vinifera as Assessed by Transcriptional Profiling

Background Ripening of fleshy fruit is a complex developmental process involving the differentiation of tissues with separate functions. During grapevine berry ripening important processes contributing to table and wine grape quality take place, some of them flesh- or skin-specific. In this study, transcriptional profiles throughout flesh and skin ripening were followed during two different seasons in a table grape cultivar ‘Muscat Hamburg’ to determine tissue-specific as well as common developmental programs. Methodology/Principal Findings Using an updated GrapeGen Affymetrix GeneChip® annotation based on grapevine 12×v1 gene predictions, 2188 differentially accumulated transcripts between flesh and skin and 2839 transcripts differentially accumulated throughout ripening in the same manner in both tissues were identified. Transcriptional profiles were dominated by changes at the beginning of veraison which affect both pericarp tissues, although frequently delayed or with lower intensity in the skin than in the flesh. Functional enrichment analysis identified the decay on biosynthetic processes, photosynthesis and transport as a major part of the program delayed in the skin. In addition, a higher number of functional categories, including several related to macromolecule transport and phenylpropanoid and lipid biosynthesis, were over-represented in transcripts accumulated to higher levels in the skin. Functional enrichment also indicated auxin, gibberellins and bHLH transcription factors to take part in the regulation of pre-veraison processes in the pericarp, whereas WRKY and C2H2 family transcription factors seems to more specifically participate in the regulation of skin and flesh ripening, respectively. Conclusions/Significance A transcriptomic analysis indicates that a large part of the ripening program is shared by both pericarp tissues despite some components are delayed in the skin. In addition, important tissue differences are present from early stages prior to the ripening onset including tissue-specific regulators. Altogether, these findings provide key elements to understand berry ripening and its differential regulation in flesh and skin.This study was financially supported by GrapeGen Project funded by Genoma España within a collaborative agreement with Genome Canada. The authors also thank The Ministerio de Ciencia e Innovacion for project BIO2008-03892 and a bilateral collaborative grant with Argentina (AR2009-0021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe