Taxonomic Identification and Molecular DNA Barcoding of Collected Wild-Growing Orchids Used Traditionally for Salep Production.

Molecular DNA barcoding combined with botanical taxonomy can be used for the identification and conservation of collected Greek orchids used for salep production as well as in the regulation of fair salep trade. A modified CTAB protocol was used for DNA extraction, amplification of barcoding regions (ITS, matK, rbcL, trnH-psbA), and sequencing. Sequencing data were assembled using Bioedit software, and the BLAST algorithm was used on the NCBI database for species identification at the genus level. Molecular barcoding data based on genetic similarity identification was in full coherence with taxonomic classification based on morphological data. The combination of ITS and matK exhibited a greater capacity to identify a species among the Greek salep samples. Out of the 53 samples examined, 52.9% were classified as Dactylorhiza spp. and 33.3% as Anacamptis spp., whereas only 6 samples were identified as Orchis spp. (11.8%). Given that a superior-quality salep beverage comes from tubers of the latter, the number of samples classified as such in northwestern Greece is unexpectedly low. A database of 53 original reference sequences from wild-growing samples of Greek origin was generated, providing a valuable resource for the identification of other salep samples from different regions. The DNA barcoding results unveiled that salep samples from northwestern Greece are related to nine members of four different genera of Orchidaceae. All species are nationally protected and covered by the CITES convention, while many of these orchids are included in the EU Directive 92/43/EEC appendix as "Other Important Species". Thus, expedited coordinated management actions are needed to ensure their survival in the future.

LED omics in Rocket Salad (Diplotaxis tenuifolia): Comparative Analysis in Different Light-Emitting Diode (LED) Spectrum and Energy Consumption.

By applying three different LED light treatments, designated as blue (B), red (R)/blue (B), red (R) and white (W) light, as well as the control, the effect on Diplotaxis tenuifolia phenotype (yield and quality), and physiological, biochemical, and molecular status, as well as growing system resource use efficiency, was examined. We observed that basic leaf characteristics, such as leaf area, leaf number, relative chlorophyll content, as well as root characteristics, such as total root length and root architecture, remained unaffected by different LEDs. Yield expressed in fresh weight was slightly lower in LED lights than in the control (1113 g m-2), with R light producing the least (679 g m-2). However, total soluble solids were significantly affected (highest, 5.5° Brix, in R light) and FRAP was improved in all LED lights (highest, 191.8 µg/g FW, in B) in comparison to the control, while the nitrate content was less (lowest, 949.2 µg/g FW, in R). Differential gene expression showed that B LED light affected more genes in comparison to R and R/B lights. Although total phenolic content was improved under all LED lights (highest, 1.05 mg/g FW, in R/B), we did not detect a significant amount of DEGs in the phenylpropanoid pathway. R light positively impacts the expression of the genes encoding for photosynthesis components. On the other hand, the positive impact of R light on SSC was possibly due to the expression of key genes being induced, such as SUS1. In summary, this research is an integrative and innovative study, where the exploration of the effect of different LED lights on rocket growing under protected cultivation, in a closed chamber cultivation system, was performed at multiple levels.

Epigenetic and Physiological Responses to Varying Root-Zone Temperatures in Greenhouse Rocket.

Greenhouse production of baby leaf vegetables grown in hydroponic floating trays has become extremely popular in recent years. Rocket (Eruca sativa Mill.) can grow in temperatures varying between 10 and 20 °C; nevertheless, a root-zone temperature (RZT) range of 18-23 °C is considered optimal for high productivity, photosynthesis, and production of metabolites. Maintaining such temperatures in winter raises production costs and prevents sustainability. In this study, we tested the impact of lower RZT on plants' status and recorded their responses while providing energy for heating using photovoltaic solar panels. We used three hydroponic tanks for cultivation; a non-heated (control) tank (12 °C) and two heated tanks; a solar panel-powered one (16 °C) and a public grid-powered one (22 °C). Methylation-sensitive amplified polymorphisms (MSAP) analysis of global methylation profiles and chlorophyll fluorescence analysis were employed to assess methylation and physiology levels of rocket leaves. We found that there is demethylation at 16 °C RZT in comparison to 22 °C RZT. Reduction of temperature at 12 °C did not reduce methylation levels further but rather increased them. Furthermore, at 16 °C, the effective quantum yield of photosystem II (PSII) photochemistry (ΦPSII) was significantly higher, with a higher PSII electron transport rate (ETR) and a significantly decreased non-regulated energy loss (ΦΝO), suggesting a better light energy use by rocket plants with higher photosynthetic performance. ΦPSII was significantly negatively correlated with DNA methylation levels. Our results show that at 16 °C RZT, where plants grow efficiently without being affected by the cold, DNA methylation and photosynthesis apparatus systems are altered. These findings corroborate previous results where hydroponic production of rocket at RZT of 16 °C is accompanied by sufficient yield showing that rocket can effectively grow in suboptimal yet sustainable root-zone temperatures.

Changes in Light Energy Utilization in Photosystem II and Reactive Oxygen Species Generation in Potato Leaves by the Pinworm Tuta absoluta.

We evaluated photosystem II (PSII) functionality in potato plants (Solanum tuberosum L.) before and after a 15 min feeding by the leaf miner Tuta absoluta using chlorophyll a fluorescence imaging analysis combined with reactive oxygen species (ROS) detection. Fifteen minutes after feeding, we observed at the feeding zone and at the whole leaf a decrease in the effective quantum yield of photosystem II (PSII) photochemistry (ΦPSII). While at the feeding zone the quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) did not change, at the whole leaf level there was a significant increase. As a result, at the feeding zone a significant increase in the quantum yield of non-regulated energy loss in PSII (ΦNO) occurred, but there was no change at the whole leaf level compared to that before feeding, indicating no change in singlet oxygen (1O2) formation. The decreased ΦPSII after feeding was due to a decreased fraction of open reaction centers (qp), since the efficiency of open PSII reaction centers to utilize the light energy (Fv'/Fm') did not differ before and after feeding. The decreased fraction of open reaction centers resulted in increased excess excitation energy (EXC) at the feeding zone and at the whole leaf level, while hydrogen peroxide (H2O2) production was detected only at the feeding zone. Although the whole leaf PSII efficiency decreased compared to that before feeding, the maximum efficiency of PSII photochemistry (Fv/Fm), and the efficiency of the water-splitting complex on the donor side of PSII (Fv/Fo), did not differ to that before feeding, thus they cannot be considered as sensitive parameters to monitor biotic stress effects. Chlorophyll fluorescence imaging analysis proved to be a good indicator to monitor even short-term impacts of insect herbivory on photosynthetic function, and among the studied parameters, the reduction status of the plastoquinone pool (qp) was the most sensitive and suitable indicator to probe photosynthetic function under biotic stress.

Vegetable Grafting From a Molecular Point of View: The Involvement of Epigenetics in Rootstock-Scion Interactions.

Vegetable grafting is extensively used today in agricultural production to control soil-borne pathogens, abiotic and biotic stresses and to improve phenotypic characteristics of the scion. Commercial vegetable grafting is currently practiced in tomato, watermelon, melon, eggplant, cucumber, and pepper. It is also regarded as a rapid alternative to the relatively slow approach of breeding for increased environmental-stress tolerance of fruit vegetables. However, even though grafting has been used for centuries, until today, there are still many issues that have not been elucidated. This review will emphasize on the important mechanisms taking place during grafting, especially the genomic interactions between grafting partners and the impact of rootstocks in scion's performance. Special emphasis will be drawn on the relation between vegetable grafting, epigenetics, and the changes in morphology and quality of the products. Recent advances in plant science such as next-generation sequencing provide new information regarding the molecular interactions between rootstock and scion. It is now evidenced that genetic exchange is happening across grafting junctions between rootstock and scion, potentially affecting grafting-mediated effects already recorded in grafted plants. Furthermore, significant changes in DNA methylation are recorded in grafted scions, suggesting that these epigenetic mechanisms could be implicated in grafting effects. In this aspect, we also discuss the process and the molecular aspects of rootstock scion communication. Finally, we provide with an extensive overview of gene expression changes recorded in grafted plants and how these are related to the phenotypic changes observed. Τhis review finally seeks to elucidate the dynamics of rootstock-scion interactions and thus stimulate more research on grafting in the future. In a future where sustainable agricultural production is the way forward, grafting could play an important role to develop products of higher yield and quality in a safe and "green" way.

Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits.

Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes.

BACKGROUND: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S.pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. RESULTS: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. CONCLUSIONS: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.

Use of the de novo transcriptome analysis of silver-leaf nightshade (Solanum elaeagnifolium) to identify gene expression changes associated with wounding and terpene biosynthesis.

BACKGROUND: Solanum elaeagnifolium, an invasive weed of the Solanaceae family, is poorly studied although it poses a significant threat to crops. Here the analysis of the transcriptome of S. elaeagnifolium is presented, as a means to explore the biology of this species and to identify genes related to its adaptation to environmental stress. One of the basic mechanisms by which plants respond to environmental stress is through the synthesis of specific secondary metabolites that protect the plant from herbivores and microorganisms, or serve as signaling molecules. One important such group of secondary metabolites are terpenes. RESULTS: By next-generation sequencing, the flower/leaf transcriptome of S. elaeagnifolium was sequenced and de novo assembled into 75,618 unigenes. Among the unigenes identified, several corresponded to genes involved in terpene biosynthesis; these included terpene synthases (TPSs) and genes of the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. Functional characterization of two of the TPSs showed that one produced the sesquiterpene (E)-caryophyllene and the second produced the monoterpene camphene. Analysis of wounded S. elaeagnifolium leaves has shown significant increase of the concentration of (E)-caryophyllene and geranyl linalool, two terpenes implicated in stress responses. The increased production of (E)-caryophyllene was matched to the induced expression of the corresponding TPS gene. Wounding also led to the increased expression of the putative 1-deoxy-D-xylulose-5-phosphate synthase 2 (DXS2) gene, a key enzyme of the MEP pathway, corroborating the overall increased output of terpene biosynthesis. CONCLUSIONS: The reported S. elaeagnifolium de novo transcriptome provides a valuable sequence database that could facilitate study of this invasive weed and contribute to our understanding of the highly diverse Solanaceae family. Analysis of genes and pathways involved in the plant's interaction with the environment will help to elucidate the mechanisms that underly the intricate features of this unique Solanum species.

The study of two barley type I-like MADS-box genes as potential targets of epigenetic regulation during seed development.

BACKGROUND: MADS-box genes constitute a large family of transcription factors functioning as key regulators of many processes during plant vegetative and reproductive development. Type II MADS-box genes have been intensively investigated and are mostly involved in vegetative and flowering development. A growing number of studies of Type I MADS-box genes in Arabidopsis, have assigned crucial roles for these genes in gamete and seed development and have demonstrated that a number of Type I MADS-box genes are epigenetically regulated by DNA methylation and histone modifications. However, reports on agronomically important cereals such as barley and wheat are scarce. RESULTS: Here we report the identification and characterization of two Type I-like MADS-box genes, from barley (Hordeum vulgare), a monocot cereal crop of high agronomic importance. Protein sequence and phylogenetic analysis showed that the putative proteins are related to Type I MADS-box proteins, and classified them in a distinct cereal clade. Significant differences in gene expression among seed developmental stages and between barley cultivars with varying seed size were revealed for both genes. One of these genes was shown to be induced by the seed development- and stress-related hormones ABA and JA whereas in situ hybridizations localized the other gene to specific endosperm sub-compartments. The genomic organization of the latter has high conservation with the cereal Type I-like MADS-box homologues and the chromosomal position of both genes is close to markers associated with seed quality traits. DNA methylation differences are present in the upstream and downstream regulatory regions of the barley Type I-like MADS-box genes in two different developmental stages and in response to ABA treatment which may be associated with gene expression differences. CONCLUSIONS: Two barley MADS-box genes were studied that are related to Type I MADS-box genes. Differential expression in different seed developmental stages as well as in barley cultivars with different seed size was evidenced for both genes. The two barley Type I MADS-box genes were found to be induced by ABA and JA. DNA methylation differences in different seed developmental stages and after exogenous application of ABA is suggestive of epigenetic regulation of gene expression. The study of barley Type I-like MADS-box genes extends our investigations of gene regulation during endosperm and seed development in a monocot crop like barley.

MASiVEdb: the Sirevirus Plant Retrotransposon Database.

BACKGROUND: Sireviruses are an ancient genus of the Copia superfamily of LTR retrotransposons, and the only one that has exclusively proliferated within plant genomes. Based on experimental data and phylogenetic analyses, Sireviruses have successfully infiltrated many branches of the plant kingdom, extensively colonizing the genomes of grass species. Notably, it was recently shown that they have been a major force in the make-up and evolution of the maize genome, where they currently occupy ~21% of the nuclear content and ~90% of the Copia population. It is highly likely, therefore, that their life dynamics have been fundamental in the genome composition and organization of a plethora of plant hosts. To assist studies into their impact on plant genome evolution and also facilitate accurate identification and annotation of transposable elements in sequencing projects, we developed MASiVEdb (Mapping and Analysis of SireVirus Elements Database), a collective and systematic resource of Sireviruses in plants. DESCRIPTION: Taking advantage of the increasing availability of plant genomic sequences, and using an updated version of MASiVE, an algorithm specifically designed to identify Sireviruses based on their highly conserved genome structure, we populated MASiVEdb (http://bat.infspire.org/databases/masivedb/) with data on 16,243 intact Sireviruses (total length >158Mb) discovered in 11 fully-sequenced plant genomes. MASiVEdb is unlike any other transposable element database, providing a multitude of highly curated and detailed information on a specific genus across its hosts, such as complete set of coordinates, insertion age, and an analytical breakdown of the structure and gene complement of each element. All data are readily available through basic and advanced query interfaces, batch retrieval, and downloadable files. A purpose-built system is also offered for detecting and visualizing similarity between user sequences and Sireviruses, as well as for coding domain discovery and phylogenetic analysis. CONCLUSION: MASiVEdb is currently the most comprehensive directory of Sireviruses, and as such complements other efforts in cataloguing plant transposable elements and elucidating their role in host genome evolution. Such insights will gradually deepen, as we plan to further improve MASiVEdb by phylogenetically mapping Sireviruses into families, by including data on fragments and solo LTRs, and by incorporating elements from newly-released genomes.

Multiple evidence for the role of an Ovate-like gene in determining fruit shape in pepper.

BACKGROUND: Grafting is a widely used technique contributing to sustainable and ecological production of many vegetables, but important fruit quality characters such as taste, aroma, texture and shape are known for years to be affected by grafting in important vegetables species including pepper. From all the characters affected, fruit shape is the most easily observed and measured. From research in tomato, fruit shape is known to be controlled by many QTLs but only few of them have larger effect on fruit shape variance. In this study we used pepper cultivars with different fruit shape to study the role of a pepper Ovate-like gene, CaOvate, which encodes a negative regulator protein that brings significant changes in tomato fruit shape. RESULTS: We successfully cloned and characterized Ovate-like genes (designated as CaOvate) from two pepper cultivars of different fruit shape, cv. "Mytilini Round" and cv. "Piperaki Long", hereafter referred to as cv. "Round" and cv. "Long" after the shape of their mature fruits. The CaOvate consensus contains a 1008-bp ORF, encodes a 335 amino-acid polypeptide, shares 63% identity with the tomato OVATE protein and exhibits high similarity with OVATE sequences from other Solanaceae species, all placed in the same protein subfamily as outlined by expert sequence analysis. No significant structural differences were detected between the CaOvate genes obtained from the two cultivars. However, relative quantitative expression analysis showed that the expression of CaOvate followed a different developmental profile between the two cultivars, being higher in cv. "Round". Furthermore, down-regulation of CaOvate through VIGS in cv. "Round" changes its fruit to a more oblong form indicating that CaOvate is indeed involved in determining fruit shape in pepper, perhaps by negatively affecting the expression of its target gene, CaGA20ox1, also studied in this work. CONCLUSIONS: Herein, we clone, characterize and study CaOvate and CaGA20ox1 genes, very likely involved in shaping pepper fruit. The oblong phenotype of the fruits in a plant of cv. "Round", where we observed a significant reduction in the expression levels of CaOvate, resembled the change in shape that takes place by grafting the round-fruited cultivar cv. "Round" onto the long-fruited pepper cultivar cv. "Long". Understanding the role of CaOvate and CaGA20ox1, as well as of other genes like Sun also involved in controlling fruit shape in Solanaceae plants like tomato, pave the way to better understand the molecular mechanisms involved in controlling fruit shape in Solanaceae plants in general, and pepper in particular, as well as the changes in fruit quality induced after grafting and perhaps the ways to mitigate them.

The study of a SPATULA-like bHLH transcription factor expressed during peach (Prunus persica) fruit development.

Extensive studies on the dry fruits of the model plant arabidopsis (Arabidopsis thaliana) have revealed various gene regulators of the development and dehiscence of the siliques. Peach pericarp is analogous to the valve tissues of the arabidopsis siliques. The stone (otherwise called pit) in drupes is formed through lignification of the fruit endocarp. The lignified endocarp in peach can be susceptible to split-pit formation under certain genetic as well as environmental factors. This phenomenon delays processing of the clingstone varieties of peach and causes economical losses for the peach fruit canning industry. The fruitfull (FUL) and shatterproof (SHP) genes are key MADS-box transcription protein coding factors that control fruit development and dehiscence in arabidopsis by promoting the expression of basic helix-loop-helix (bHLH) transcription factors like Spatula (SPT) and Alcatraz (ALC). Results from our previous studies on peach suggested that temporal regulation of PPERFUL and PPERSHP gene expression may be involved in the regulation of endocarp margin development. In the present study a PPERSPATULA-like (PPERSPT) gene was cloned and characterized. Comparative analysis of temporal regulation of PPERSPT gene expression during pit hardening in a resistant and a susceptible to split-pit variety, suggests that this gene adds one more component to the genes network that controls endocarp margins development in peach. Taking into consideration that no ALC-like genes have been identified in any dicot plant species outside the Brassicaceae family, where arabidopsis belongs, PPERSPT may have additional role(s) in peach that are fulfilled in arabidopsis by ALC.