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

<p>Abstract</p> <p>Background</p> <p>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 <it>Ovate</it>-like gene, <it>CaOvate</it>, which encodes a negative regulator protein that brings significant changes in tomato fruit shape.</p> <p>Results</p> <p>We successfully cloned and characterized <it>Ovate</it>-like genes (designated as <it>CaOvate</it>) 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 <it>CaOvate </it>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 <it>CaOvate </it>genes obtained from the two cultivars. However, relative quantitative expression analysis showed that the expression of <it>CaOvate </it>followed a different developmental profile between the two cultivars, being higher in cv. "Round". Furthermore, down-regulation of <it>CaOvate </it>through VIGS in cv. "Round" changes its fruit to a more oblong form indicating that <it>CaOvate </it>is indeed involved in determining fruit shape in pepper, perhaps by negatively affecting the expression of its target gene, <it>CaGA20ox1</it>, also studied in this work.</p> <p>Conclusions</p> <p>Herein, we clone, characterize and study <it>CaOvate </it>and <it>CaGA20ox1 </it>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 <it>CaOvate</it>, 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 <it>CaOvate </it>and <it>CaGA20ox1</it>, as well as of other genes like <it>Sun </it>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.</p

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.General Secretariat of Research and Technology (GSRT) {[}09-SYN-23-879]; grant SEE-ERA. NET PLUS {[}ERA 64/01]; grant KRIPIS {[}MIS 448840

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 &deg;C; nevertheless, a root-zone temperature (RZT) range of 18&ndash;23 &deg;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&rsquo; 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 &deg;C) and two heated tanks; a solar panel-powered one (16 &deg;C) and a public grid-powered one (22 &deg;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 &deg;C RZT in comparison to 22 &deg;C RZT. Reduction of temperature at 12 &deg;C did not reduce methylation levels further but rather increased them. Furthermore, at 16 &deg;C, the effective quantum yield of photosystem II (PSII) photochemistry (&Phi;PSII) was significantly higher, with a higher PSII electron transport rate (ETR) and a significantly decreased non-regulated energy loss (&Phi;&Nu;O), suggesting a better light energy use by rocket plants with higher photosynthetic performance. &Phi;PSII was significantly negatively correlated with DNA methylation levels. Our results show that at 16 &deg;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 &deg;C is accompanied by sufficient yield showing that rocket can effectively grow in suboptimal yet sustainable root-zone temperatures

Study of the molecular mechanisms that control fruit shape in pepper (Capsicun annuum), in relation to grafting

An important fruit quality characteristic in vegetables and especially in pepper is shape and so the study of the molecular mechanisms and other factors that possibly affect this characteristic, is important. Grafting is one such factor that often influences scion fruit shape in some rootstock-scion combinations. In IN.A.B. lab it was found that grafting of two pepper cultivars of different fruit shape, cv. Round on cv. Long, changed the resulting grafted plant fruits shape (T1 generation) that instead of being round (as the cv. Round scion) became oblong. The change in fruit shape was stably inherited in the next T2 generation that originated from the changed fruits of the T1 generation. These results have been the starting point of this study. ISSR marker analysis has showed that changes are detected in the T2 progenies genetic profile in comparison to the genetic profile of cv. Round (the scion) though no epigenetic differences were detected in T2 progenies epigenetic profile as long as in cv. Round και cv. Long. All these findings suggest that probably genetic changes have occurred during grafting and inherited to the progenies. In particular, genes CaOvate and CaGA20ox1 were shown to be involved in determining fruit shape in pepper according to what it is known, CaOvate acting as a negative regulator of CaGA20ox1. The expression of Cafw2.2 is lower in a large-fruited pepper cultivar of “bell type” in comparison to the expression in the small-fruited cv. Round, in the 10 days after anthesis fruits. Gene CaSun-like is not involved in determining pepper fruit shape. Differential display experiments have shown that genes are differentially expressed in T2 progenies compared to cv. Round. Genes CaOvate and CaSun-like are expressed differently in the T2 progenies in comparison to cv. Round and cv. Long. Another gene, designated as 5A, is expressed 11 times more in the T2 progenies in comparison to cv. Round.In order to explore deeply the causes of the graft-induced changes in pepper fruit shape inherited in the T2 generation and the grafting changes in fruit quality in general, conserved miRNAs were studied in grafted pepper plants since miRNAs are epigenetic switches that control the expression of many target-genes and recently were found to be induced or suspended by grafting. The study of conserved miRNAs showed that the expression of the miRNAs does not change in grafted cv. Round on cv. Long plants (L/R). However the study of a gene that codes for protein DCL4 that is involved in the production of mobile small RNAs inside plants, was found to be down-regulated in two L(rootstock)/R(scion) grafted pepper plants in comparison to the WT cv. Round non grafted plants.Ένα σημαντικό ποιοτικό χαρακτηριστικό στα λαχανικά και ιδιαίτερα στην πιπεριά είναι το σχήμα του καρπού με συνέπεια η μελέτη των μοριακών μηχανισμών και άλλων παραγόντων που πιθανόν το επηρεάζουν να είναι σημαντική. Ένας τέτοιος παράγοντας είναι ο εμβολιασμός που συχνά έχει επίπτωση στο σχήμα του καρπού του εμβολίου, σε ορισμένους συνδυασμούς εμβολιαζόμενων γενοτύπων. Στο εργαστήριο του ΙΝΕΒ βρέθηκε ότι ο εμβολιασμός δύο ποικιλιών με διαφορετικό σχήμα καρπού, της cv. Round (εμβόλιο) επί της cv. Long (υποκείμενο) προκάλεσε αλλαγή στο σχήμα των καρπών του εμβολίου (Τ1 γενεά), από το στρογγυλό προς το επίμηκες. H αλλαγή αυτή κληρονομήθηκε σταθερά και στους απογόνους της Τ2 γενεάς που προήλθαν από τους καρπούς της T1 γενεάς που διαφοροποιήθηκαν ως προς το σχήμα. Τα αποτελέσματα αυτά αποτέλεσαν την αφετηρία για την παρούσα ερευνητική εργασία. Ανάλυση ISSR δεικτών έδειξε ότι καταγράφονται αλλαγές στο γενετικό προφίλ των Τ2 απογόνων σε σχέση με το εμβόλιο, cv. Round, ενώ δεν καταγράφονται διαφορές στο επιγενετικό προφίλ των T2 απογόνων και στις δύο ποικιλίες, cv. Round και cv. Long. Τα ευρήματα αυτά δείχνουν ότι έχουν συμβεί κατά τον εμβολιασμό γενετικές αλλαγές και μάλιστα αυτές έχουν κληρονομηθεί στους απογόνους. Ειδικότερα, τα γονίδια CaOvate και CaGA20ox1 βρέθηκε ότι εμπλέκονται στον καθορισμό του σχήματος του καρπού της πιπεριάς σύμφωνα με ότι είναι γνωστό, δρώντας το ένα ως αρνητικός ρυθμιστής του άλλου. Η έκφραση του Cafw2.2-like είναι μικρότερη σε μία μεγαλόκαρπη ποικιλία πιπεριάς τύπου «καμπάνα» σε σχέση με την μικρόκαρπη cv. Round, στους καρπούς των 10 ημερών μετά την άνθηση. Το γονίδιο CaSun-like δεν φαίνεται να συνδέεται με το σχήμα. Στους Τ2 απογόνους του εμβολιασμού με την κληρονομούμενη αλλαγή στο σχήμα των καρπών, καταγράφηκε διαφορική έκφραση γονιδίων σε σχέση με το υποκείμενο, cv. Round. Τα CaOvate και CaSun-like εκφράζονται διαφορετικά στους Τ2 απογόνους σε σχέση με τις cv. Round και cv. Long. Ακόμα ένα γονίδιο, το γονίδιο που ονομάστηκε 5Α, εκφράζεται 11 φορές περισσότερο στους Τ2 απογόνους σε σχέση με την cv. Round. Προκειμένου να εξερευνηθούν περισσότερο οι μηχανισμοί που προκαλούν την αλλαγή στο σχήμα του καρπού που κληρονομήθηκε στους Τ2 απογόνους αλλά και γενικότερα τις αλλαγές στην ποιότητα του καρπού κατά τον εμβολιασμό, μελετήθηκαν συντηρημένα miRNAs που ως επιγενετικοί ρυθμιστές επηρεάζουν την έκφραση πολλών γονιδίων και έχουν βρεθεί να επάγονται ή αναστέλλονται κατά τον εμβολιασμό. Η μελέτη των συντηρημένων miRNAs που βρέθηκαν μετά από εκτεταμένες βιοπληροφορικές αναλύσεις έδειξε ότι δεν αλλάζει η έκφρασή τους κατά τον εμβολιασμό της cv. Round στην cv. Long. Ωστόσο ένα γονίδιο που κωδικοποιεί την πρωτεΐνη DCL4, κύριο μεταφορέα του σήματος της γονιδιακής αποσιώπησης που συνδέεται με τα μικρά RNAs στα φυτά, βρέθηκε να εκφράζεται λιγότερο στα εμβολιασμένα φυτά της cv. Round επί της cv. Long σε σχέση με τον αυτόρριζο μάρτυρα, την cv. Round

MASiVEdb: the Sirevirus Plant Retrotransposon Database

<p>Abstract</p> <p>Background</p> <p>Sireviruses are an ancient genus of the <it>Copia</it> 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 <it>Copia</it> 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.</p> <p>Description</p> <p>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 (<url>http://bat.infspire.org/databases/masivedb/</url>) 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.</p> <p>Conclusion</p> <p>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.</p

Taxonomic and molecular characterization of 15 wild-growing tulip species of Greece using the internal transcribed spacer (ITS) nuclear marker in combination with the psbA-trnH and trnL/trnF plastid markers

AbstractDue to the absence or scarcity of DNA sequence data in GenBank regarding Mediterranean, Balkan or Greek native tulip species, we performed molecular characterization of the 15 wild-growing Greek tulip species through analysis of the internal transcribed spacer nrDNA and the psbA/trnH and trnL/trnF cpDNA regions. To facilitate conservation and sustainable utilization efforts, the aim was to identify and complement the genetic distinctiveness of six Greek endemic and five Balkan or Aegean endemic and/or subendemic wild-growing Greek native tulips and two naturalized ones of Asiatic origin, among which seven are threatened with extinction. Molecular phylogenetic analysis of 148 DNA sequences was conducted, and 85 Tulipa taxa (species and subspecies) were aligned and compared to 40 new DNA sequences generated herein for the 15 wild-growing Greek tulips. These three molecular markers resulted in 354 single-nucleotide polymorphisms (SNPs) and 20 distinct indels for all Tulipa taxa, of which 100 SNPs and 12 indels were related to the Greek tulips. Phylogenetic dendrograms were constructed for all the above-mentioned molecular markers, and the DNA sequences were analyzed separately and jointly, leading to three distinct clusters for the 15 wild-growing Greek tulips. Group 1 included Greek members of subgenus Tulipa, that is, T. scardica, T. undulatifolia, T. rhodopea, T. agenensis and T. raddii; Group 2 included only T. clusiana (subgenus Clusianae) and Group 3 included Greek members of subgenus Eriostemones, that is, *T. bakeri, T. saxatilis, T. australis, *T. cretica, *T. goulimyi, *T. orphanidea T. bithynica, *T. hageri and *T. doerfleri (asterisks indicate single-country endemics)

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

The Influence of Water Nitrate Concentration Combined with Elevated Temperature on Rainbow Trout <i>Oncorhynchus mykiss</i> in an Experimental Aquaponic Setup

Intensive recirculating systems are a fast-developing sector of aquaculture. While several warm-water fish have been reared in aquaponics, almost no data are available for cold-water species. The determination of nitrate toxicity thresholds in recirculating aquaculture is crucial. Different pollutants are typically more toxic at elevated temperatures. We investigated the performance of Oncorhynchus mykiss under two different nitrate levels and two temperatures. We applied a 2 × 2 factorial design, where fish (9.78 ± 0.51 g) were exposed to nitrate concentrations of 40 or 110 mg/L NO3− and to temperatures of 17 °C or 21 °C for 20 days. This study focused on understanding the physiological responses of rainbow trout to relatively low nitrate levels under heat stress in order to investigate the feasibility of integrating this species into commercial aquaponics. The growth, condition, and expression of genes involved in metabolism, heat shock, antioxidant, and immune response were assessed in the liver, together with the activities of enzymes related to glucose and fatty acid metabolism. High nitrate levels at 17 °C affected the condition but did not alter growth, leading to increased glycolytic potential and, occasionally, a greater reliance on lipid oxidation. Antioxidant defense was mainly induced due to high nitrates and the similar expression patterns of antioxidant genes observed under high nitrate at both 17 °C and 21 °C. Warm exposure decreased condition and growth, leading to greatly reduced glucokinase transcription, irrespective of the nitrate levels. Exposure to 21 °C and high nitrate led to equivalent growth and condition as well as to a milder inflammatory response combined with metabolic readjustments (enhancement of glycolytic and lipid oxidation pathways) compared to the low nitrates at 21 °C. Based on the results, rearing at a temperature close to 21 °C should be avoided for fingerling growth, while NO3− concentration until 110 mg/L may not have severe impacts on fingerling health and growth at 17 °C. In addition, rainbow trout fingerlings can tolerate a 20-day exposure at 21 °C and NO3− up to 110 mg/L. Additional factors should always be considered, such as specific water quality parameters, for a comprehensive approach to assessing the feasibility of rainbow trout aquaculture in aquaponics

Durum Wheat Breeding in the Mediterranean Region: Current Status and Future Prospects

This brief historical review focuses on durum wheat domestication and breeding in the Mediterranean region. Important milestones in durum wheat breeding programs across the countries of the Mediterranean basin before and after the Green Revolution are discussed. Additionally, the main achievements of the classical breeding methodology are presented using a comparison of old and new cultivars. Furthermore, current breeding goals and challenges are analyzed. An overview of classical breeding methods in combination with current molecular techniques and tools for cultivar development is presented. Important issues of seed quality are outlined, focusing on protein and characteristics that affect human health and are connected with the consumption of wheat end-products