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The stilbene synthase multigenic family in grapevine: gemome-wide analysis and transcriptional regulation
No full textPlant stilbenes represent a relatively small group of phenylpropanoid compounds
characterized by a diphenylethylene backbone and have been detected in only a few
unrelated plant species, including pine (Pinaceae), peanut (Fabaceae), sorghum (Poaceae)
and grapevine (Vitaceae) (Morales et al., 2000). As with other phenylpropanoids, stilbenes
accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C
exposure and treatment with chemicals (Dixon and Paiva 1995). During the last decade
stilbenes, and resveratrol in particular, have captured the attention of biology and medicine
due to both the biological and medicinal activities of those compounds. Among the
biological effects they have been suggested to act as deterrents against animals and insects
(Torres et al. 2003), antifungal compounds (Morales et al., 2000; Jeandet et al., 2002),
allochemicals (Seigler 2006; Fiorentino et al., 2008) and antioxidants (Privat et al., 2002).
In terms of medical applications, they have been shown to have beneficial effects in the
treatment of cardiovascular disease, cancer, diabetes and neurodegenerative diseases (Baur
et al., 2006).
Stilbenes are formed via the phenylalanine/polymalonate route (Hall and Yu, 2008). The
last step of the biosynthetic pathway is catalysed by stilbene synthase (STS), which
produces resveratrol in a single enzymatic reaction utilizing p-coumaryl-CoA and three
malonyl-CoA units as substrates (Schröder and Schröder 1990). Despite the fact that all
higher plants are able to accumulate basic compounds like p-coumaroyl CoA or malonyl-
CoA through general and ubiquitous enzymes such as phenylalanine ammonia lyase (PAL),
cinnamate 4-hydroxylase (C4H) or 4-coumaroyl–CoA ligase (4CL), only a limited group of
plants are able to produce resveratrol (and derivatives) through the STS enzyme. This
stilbene-producing enzyme belongs to the large CHS type III polyketide synthase family,
the main members of which are the chalcone synthases, which shares 75-90% of the amino
acid sequence with STSs (Schröder et al., 1988). Both of these enzymes utilize the same
substrate, but through different cyclization events lead on one hand to the production of
chalcones and flavonoids and, on the other, to the production of resveratrol and stilbenes.
Some plant species, such as Pine, Fallopia japonica (syn. Polygonum cuspidatum) and grapevine during several stages of berry development, constitutively accumulate large
amounts of stilbenes (Beñová et al., 2008). However, most studies concerning stilbene
accumulation have been conducted on tissues of peanut and grapevine, where they
accumulate in response to various biotic and abiotic stresses, as a result of increased
transcription of both STS genes and upstream enzymes in the phenylpropanoid pathway
such as PAL and C4H (Lanz et al., 1990; Bais et al., 2000). While little is known about the
transcriptional regulation of the stilbene biosynthetic pathway, a number of studies have
demonstrated a role for transcription factors in the regulation of other steps of the
phenylpropanoid pathway. These regulators include R2R3-MYB transcription factors (TFs),
responsible for the regulation of flavonols, lignin and anthocyanin metabolism (Boudet
2007). The R2R3-MYB TF group is the largest MYB TF sub-family in plants (Du et al.,
2009) with the grapevine genome estimated to contain 108 R2R3-MYB members (Matus et
al., 2008). To date, most R2R3-MYBs have been reported to play a major role in the
regulation of secondary metabolism, such as the phenylpropanoid biosynthesis. In
grapevine, R2R3-MYB factors have been demonstrated to be involved in the regulation of
several steps of the flavonoid biosynthetic pathway: VvMYBA1 and VvMYBA2 are involved
in the regulation of anthocyanin biosynthesis. VvMYB5a, VvMYB5b and VvMYBPA1 appear
to control general branches of the flavonoid pathway and VvMYB12 regulates the
production of flavonols (Bogs et al., 2007; Walker et al., 2007; Deluc et al., 2008).
This study involved two principal components: (a) the genome-wide analysis of the whole
STS multigenic family in grape and (b) the identification of candidate transcription factors
involved in the regulation of the grape stilbene synthase pathway. The first point was
achieved firstly by the identification, annotation and phylogenetic study of all members
predicted to belong to the STS family and secondarily by transcriptional analysis of the
whole STS family in grapevine in response to biotic and abiotic stress conditions and in
unstressed healthy tissues at different developmental stages (in collaboration with the
University of Verona). The expression of all identified VvSTS members predicted on the
12X V1 grape genome draft was evaluated using mRNA sequencing technology on Pinot
noir leaf discs treated by wounding, exposure to UV-C light and downy mildew infection.
The analysis was performed using a next generation whole-transcriptome sequencing
technology (Illumina) and revealed different sub-groups of VvSTS genes characterized by different degrees of response to the different elicitors. The constitutive expression of VvSTS
genes in different tissues was analysed with a different approach, in collaboration with the
University of Verona. A grapevine expression atlas obtained using a Nimblegen and
Combimatrix microarray technology based on the 12X V1 coverage assembly predictions
(kindly provided by the University of Verona) was screened for VvSTS genes providing
interesting insights into the transcriptional regulation of VvSTS genes in grapevine. A wide
range of grapevine tissues were analysed, including leaf, berry tissues, such as exocarp,
endocarp and seed, in vitro roots, rachis, stem, tendril etc. Moreover, tissues were evaluated
at different developmental stages such as fruit set, pre-ripening, ripening or withering
concerning the berry development or at different temporal stages regarding leaves or other
vegetative tissues. Analyses using the grapevine expression atlas confirmed the existence of
different VvSTS subgroups characterized by different expression patterns and provided a
comprehensive picture of VvSTS expression patterns in planta. Considering the close
relationship between the flavonoid and stilbene biosynthetic pathways and the fact that
certain key genes within the flavonoid pathway have been shown to regulated by R2R3-
MYB transcription factors (Bogs et al. 2007; Walker et al. 2007; Deluc et al. 2008) the
mRNAseq data obtained from stresses grape tissues were also examined for the expression
MYB TFs that might show similar expression patterns to the VvSTS genes. Of the 108
grape R2R3-MYB factors analysed, two accessions displayed similar expression patterns to
the inducible VvSTS genes. These two accessions were previously designated VvMYB14
and VvMYB15 by Matus et al. (2008) based on their homology to the Arabidopsis thaliana
MYB14 gene. Validation of the expression data obtained from the mRNAseq analysis, was
achieved using a quantitative RT-PCR approach, by screening in more detail the
relationship between selected VvSTS and VvMYB14 expression patterns in grape tissues
following the application of abiotic and biotic stress treatments. Two highly responsive
VvSTS genes were selected for analysis, VvSTS22 and VvSTS36. All treatments confirmed a
strong correlation between the expression of VvMYB14 and the two VvSTS genes. To obtain
direct evidence of the regulation of VvSTS promoter activity by VvMYB14 a gene reporter
assay using a dual luciferase assay system was utilized. Chardonnay cell suspensions
transiently expressing VvSTS promoter-luciferase expression constructs showed a
statistically significant increase of VvSTS promoter activity when co-transformed with VvMYB14. Finally, to validate the role of VvMYB14 in the regulation of the VvSTS pathway
in planta, attempts were made to silence VvMYB14 in a grapevine hairy root system. In a
preliminary screening of transformed hairy root lines, those lines showing the highest levels
of VvMYB14 silencing were found to show the lowest induction of VvSTS36 in response to
wounding, giving a first biological confirmation of real role for VvMYB14 in the regulation
of the VvSTS pathway.Gli stilbeni rappresentano un gruppo minore di fenilpropanoidi, caratterizzati da una
matrice chimica difenil-etilenica, rilevabili solamente in un ristretto gruppo di piante
superiori tra cui alcune specie di pino (Pinaceae), arachide (Fabaceae), Sorgo (Poaceae) e
vite (Vitacea) (Morales et al., 2000). Come avviene per altri fenilpropanoidi, ad es. nel caso
dei flavonoidi, l’accumulo di stilbeni è legato per lo più all’esposizione della pianta a stress
di tipo biotico e non, tra i quali danneggiamento meccanico, esposizione a radiazione
ultravioletta (UV-C), infezione e trattamento con sostanze chimiche (Dixon and Paiva
1995). Nell’arco degli ultimi decenni gli stilbeni, e il resveratrolo in particolare, hanno
catalizzato l’attenzione di numerosi studi scientifici, in virtù delle loro proprietà biologiche
e mediche. Dal punto di vista biologico, queste sostanze possiedono svariate funzioni,
fungendo da deterrenti contro erbivori e insetti (Torres et al. 2003), da composti ad azione
anti fungina (Morales et al., 2000; Jeandet et al., 2002), da sostanze allelopatiche (Seigler
2006; Fiorentino et al., 2008) e da antiossidanti (Privat et al., 2002). Dal punto di vista
medico, sembrano invece coinvolti in un generale allungamento delle prospettive di vita,
rivestendo un ruolo importante nella prevenzione e cura di malattie cardio-respiratorie,
neuro-degenerative ed altre malattie quaili cancro, diabete, ecc... (Baur et al., 2006).
La pathway di sintesi degli stilbeni può essere considerata una pathway “alternativa” a
quella che sovrintende alla biosintesi dei flavonoidi. Tutte le piante superiori sono in grado
di produrre composti quali il p-cumaroyl CoA o il cinnamoyl CoA, attraverso l’azione di
enzimi ubiquitari nel regno vegetale quali la fenilanina ammonia liasi (PAL), la cinnamato
4-idrossilasi (C4H) o la 4-cumaroil–CoA ligasi (4CL). Questi composti rappresentano il
substrato di partenza per la chalcone sintasi (CHS), enzima chiave che porta allo scaffold di
sintesi dei flavonoidi. Soltanto in quelle piante in grado di produrre e accumulare stilbeni
queste molecole fungono da substrato anche per le stilbene sintasi (STS), una classe di
enzimi strettamente correlate alle chalcone sintasi, responsabili della biosintesi di
resveratrolo e degli stilbeni in generale. Le STSs appartengono alla superfamiglia delle
poliketide sintasi del tipo III, di cui la CHS rappresenta il principale esponente. Alcune
specie vegetali, tra cui pino (Pinus spp.), Fallopia japonica (Polygonum cuspidatum) o vite
(Vitis vinifera) in alcuni stadi di sviluppo della bacca, sono in grado di accumulare queste molecole in modo costitutivo (Beñová et al., 2008). Nonostante ciò, gran parte degli studi
sull’accumulo di stilbeni è stata condotta in arachide o vite, piante nelle quali la presenza di
tali composti è prevalentemente legata alla risposta a stress di tipo biotico e abiotico, come
risultato dell’attivazione trascrizionale delle STSs e di geni codificanti per enzimi a monte
nella pathway generale dei fenilpropanoidi quali PAL o C4H (Lanz et al., 1990; Bais et al.,
2000).
A oggi, non esiste alcuna informazione riguardo a fattori di trascrizione (TFs) coinvolti
nella regolazione di questa pathway, tuttavia, nel corso degli ultimi anni, un numero sempre
maggiore di informazioni si è accumulato per ciò che concerne la regolazione della
pathway dei flavonoidi. In tale contesto, un ruolo fondamentale sembra ascrivibile alla
sottofamiglia dei fattori di trascrizione MYB-R2R3, responsabili della regolazione del
metabolismo dei flavonoli, lignine e antocianine (Boudet 2007). Il gruppo dei MYB-R2R3
rappresenta una delle sotto-famiglie di TFs più numerose nel regno vegetale, con ben 108
membri identificati da una recente analisi della copertura 8.4X del genoma di vite (Matus et
al., 2008). In vite, la caratterizzazione funzionale di alcuni MYB-R2R3 ha mostrato il loro
coinvolgimento in diversi steps della biosintesi dei flavonoidi: VvMYBA1 e VvMYBA2 sono
ad esempio coinvolti nella regolazione delle sintesi di antocianine, VvMYB5A, VvMYB5B e
VvMYBPA1 regolano diversi geni strutturali della pathway dei flavonoidi, mentre
VvMYB12 sembra coinvolto nella sintesi di flavononi (Bogs et al., 2007; Walker et al.,
2007; Deluc et al., 2008). L’obbiettivo di questo studio si è articolato in due
macroargomenti principali: la caratterizzazione genomica e trascrizionale dell’intera
famiglia multigenica delle stilbene sintasi in vite e l’individuazione, con conseguente
caratterizzazione funzionale, di fattori di trascrizione coinvolti nella sua regolazione. La
caratterizzazione genomica dell’intera famiglia delle stilbene sintasi in vite si è sviluppata
dapprima mediante l’identificazione, l’annotazione e lo studio delle relazioni filogenetiche
che intercorrono tra i diversi membri di tale famiglia e, in seguito, valutando il profilo di
espressione di ogni singolo gene in diversi organi in vari stadi di sviluppo e in dischi
fogliari sottoposti a stress di varia natura. In quest’ultimo caso, l’espressione dei membri
identificati nel genoma con copertura 12X V1 è stata saggiata tramite analisi dell’intero
trascrittoma (mRNA-seq) usando una tecnologia di sequenziamento di nuova generazione
(NGS) Illumina e rilevando l’esistenza di diversi gruppi di STSs caratterizzati da gradi diversi di responsività ai vari stress. L’analisi di espressione in diversi organi e stadi di
sviluppo è stata invece realizzata in collaborazione con l’Università di Verona, che ha
fornito un atlante di espressione basato su tecnologia microarray di tipo Nimblegen e
Combimatrix. Le sonde presenti su tali chip sono state disegnate sulla base delle predizioni
ottenute dalla copertura 12X V1 del genoma ottenuto dalla linea PN40024 e ibridate con
numerosi organi di vite, tra cui foglia, bacca divisa in esocarpo, endocarpo e seme, radici in
vitro, germoglio, tendrile ecc...Tali tessuti sono stati considerati in diversi stadi temporali
tra i quali allegagione, invaiatura, pre-maturazione, maturazione e appassimento per ciò che
riguarda bacca o stadi giovanili o senescenti per foglia. In questo caso le analisi hanno
parzialmente validato i dati ottenuti nell’analisi mRNA-seq oltre a fornire un quadro
dettagliato del pattern di espressione di questi geni nell’intero sistema pianta in assenza di
stress.
Il secondo scopo del lavoro di seguito presentato è stato l’identificazione di fattori di
trascrizione coinvolti nella regolazione della pathway biosintetica responsabile della sintesi
di stilbeni. Data la stretta relazione che intercorre tra la pathway di sintesi degli stilbeni e
quella dei flavonoidi, e sulla base delle numerose evidenze scientifiche che legano la
regolazione di quest’ultima alla attività di MYB-R2R3, (Deluc et al. 2008; Bogs et al. 2007;
Walker et al. 2007), i dati Illumina ottenuti dai tessuti stressati sono stati analizzati con lo
scopo di individuare geni MYB-R2R3 co-espressi con le VvSTS. Di 108 MYB-R2R3
analizzati, soltanto due geni hanno evidenziato un pattern di espressione correlato a quello
delle VvSTS. Tali geni sono già stati annotati da Matus et al. (2008) e prendono il nome di
VvMYB14 e VvMYB15. Successive analisi real time su dischi fogliari sottoposti a stress
biotici e non, hanno confermato una marcata co-espressione tra VvMYB14 in particolare e
due VvSTS altamente stress-inducibili (VvSTS22 e VvSTS36). Tramite saggio “gene
reporter” è stata validata una reale interazione tra VvMYB14 e le sequenze promotoriali di
queste due stilbene sintasi e, al fine di confermare il ruolo di VvMYB14 anche in planta,
sono state create delle linee silenziate di Hairy Roots. In uno screening preliminare delle
linee trasformate, quelle che mostrano il più alto grado di silenziamento di VvMYB14 sono
anche quelle che mostrano il più basso grado di induzione di VvSTS36, confermando un
ruolo di questo fattore di trascrizione nella regolazione trascrizionale della pathway
biosintetica delle stilbene sintasi
Trends in Apomixis Research: The 10 Most Cited Research Articles Published in the Pregenomic and Genomic Eras
Get PDFApomixis, or asexual reproduction by seed, represents an easy shortcut for life cycle renewal based on maternal embryo production without ploidy reduction (meiosis) and ploidy restitution (syngamy). Although the first studies officially published on this topic in scientific journals date back to the early 1930s, the identification and introduction of genes involved in asexual reproduction in species of agronomic interest still represent a major challenge. Through a bibliometric analysis of the research programs implemented in apomixis over the last 40 years, the present study was aimed to discuss not only the main findings achieved but also the investigational methods and model species used. We split the critical survey of the most cited original articles into pregenomic and genomic eras to identify potential trends and depict scenarios that have emerged in the scientific community working on apomixis, as well as to determine any correlation between the exponential increase in acquired basic knowledge and the development of advanced analytical technologies. This review found a substantial stagnation in the use of the same model species, with few exceptions, for at least 40 years. In contrast, the development of new molecular techniques, genomic platforms, and repositories has directly affected the approaches used in research, which has been directed toward an increasingly focused study of the genetic and epigenetic determinants of apomixis
Genomic characterization and expression analysis of genes belonging to a family of ABC transporters involved in response to stress in Vitis vinifera L.
No full textThe leaf transcriptome of fennel (Foeniculum vulgare Mill.) enables characterization of the t-anethole pathway and the discovery of microsatellites and single-nucleotide variants
Get PDFFennel is a plant species of both agronomic and pharmaceutical interest that is characterized by a shortage of genetic and molecular data. Taking advantage of NGS technology, we sequenced and annotated the first fennel leaf transcriptome using material from four different lines and two different bioinformatic approaches: de novo and genome-guided transcriptome assembly. A reference transcriptome for assembly was produced by combining these two approaches. Among the 79,263 transcripts obtained, 47,775 were annotated using BLASTX analysis performed against the NR protein database subset with 11,853 transcripts representing putative full-length CDS. Bioinformatic analyses revealed 1,011 transcripts encoding transcription factors, mainly from the BHLH, MYB-related, C2H2, MYB, and ERF families, and 6,411 EST-SSR regions. Single-nucleotide variants of SNPs and indels were identified among the 8 samples at a frequency of 0.5 and 0.04 variants per Kb, respectively. Finally, the assembled transcripts were screened to identify genes related to the biosynthesis of t-anethole, a compound well-known for its nutraceutical and medical properties. For each of the 11 genes encoding structural enzymes in the t-anethole biosynthetic pathway, we identified at least one transcript showing a significant match. Overall, our work represents a treasure trove of information exploitable both for marker-assisted breeding and for in-depth studies on thousands of genes, including those involved in t-anethole biosynthesis
cpDNA barcoding by combined End-Point and Real-Time PCR analyses to identify and quantify the main contaminants of oregano (Origanum vulgare L.) in commercial batches
Get PDFOregano (Origanum vulgare L.) is a flowering plant that belongs to the mint family (Lamiaceae). It is used as a culinary herb and is often commercialized as a fine powder or a mixture of small fragments of dried leaves, which makes morphological recognition difficult. Like other commercial preparations of drugs and spices, the contamination of oregano mixtures with vegetable matter of lower quality, or the use of generic misleading names, are frequent and stress the need to develop a molecular traceability system to easily, quickly, and cheaply unveil these scams. The DNA-based analytical approach known as cpDNA barcoding is particularly suited for fraud identification in crop plant species (fresh products and food derivatives), and it represents a promising traceability tool as an alternative or complement to traditional detection methods. In the present study, we used a combined approach based on both qualitative and quantitative cpDNA barcoding with end-point and real-time polymerase chain reaction (PCR) analyses to assess the type and degree of contamination in commercial batches of common oregano. In a preliminary qualitative screening, we amplified, cloned, and sequenced a number of universal trnH-psbA- and trnL-barcoded regions, to identify the main contaminants in the samples under investigation. On the basis of these findings, we then developed and validated a species-specific and sequence-targeted method of testing for the quantitative assessment of contaminants, using trnL gene intron assays. Surprisingly, the results obtained in our case study indicated an almost total absence of O. vulgare in the commercial batches analyzed, but a high presence of group I contaminants (Satureja pilosa Velen.), and a moderate presence of group II contaminants (Cistus lanidifer L./Cistus albidus)
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The Mitochondrial Genome Assembly of Fennel (Foeniculum vulgare) Reveals Two Different atp6 Gene Sequences in Cytoplasmic Male Sterile Accessions
No full textCytoplasmic male sterility (CMS) has always aroused interest among researchers and breeders, being a valuable resource widely exploited not only to breed F1 hybrid varieties but also to investigate genes that control stamen and pollen development. With the aim of identifying candidate genes for CMS in fennel, we adopted an effective strategy relying on the comparison between mitochondrial genomes (mtDNA) of both fertile and sterile genotypes. mtDNA raw reads derived from a CMS genotype were assembled in a single molecule (296,483 bp), while a draft mtDNA assembly (166,124 nucleotides, 94 contigs) was performed using male fertile sample (MF) sequences. From their annotation and alignment, two atp6-like sequences were identified. atp6-, the putative mutant copy with a 300 bp truncation at the 5'-end, was found only in the mtDNA of CMS samples, while the wild type copy (atp6 +) was detected only in the MF mtDNA. Further analyses (i.e., reads mapping and Sanger sequencing), revealed an atp6 + copy also in CMS samples, probably in the nuclear DNA. However, qPCRs performed on different tissues proved that, despite its availability, atp6 + is expressed only in MF samples, while apt6-mRNA was always detected in CMS individuals. In the light of these findings, the energy deficiency model could explain the pollen deficiency observed in male sterile flower. atp6-could represent a gene whose mRNA is translated into a not-fully functional protein leading to suboptimal ATP production that guarantees essential cellular processes but not a high energy demand process such as pollen development. Our study provides novel insights into the fennel mtDNA genome and its atp6 genes, and paves the way for further studies aimed at understanding their functional roles in the determination of male sterility
Genomics of flower identity in grapevine (Vitis vinifera L.)
Get PDFThe identity of the four characteristic whorls of typical eudicots, namely, sepals, petals, stamens, and carpels, is specified by the overlapping action of homeotic genes, whose single and combined contributions have been described in detail in the so-called ABCDE model. Continuous species-specific refinements and translations resulted in this model providing the basis for understanding the genetic and molecular mechanisms of flower development in model organisms, such as Arabidopsis thaliana and other main plant species. Although grapevine (Vitis vinifera L.) represents an extremely important cultivated fruit crop globally, studies related to the genetic determinism of flower development are still rare, probably because of the limited interest in sexual reproduction in a plant that is predominantly propagated asexually. Nonetheless, several studies have identified and functionally characterized some ABCDE orthologs in grapevine. The present study is intended to provide a comprehensive screenshot of the transcriptional behavior of 18 representative grapevine ABCDE genes encoding MADS-box transcription factors in a developmental kinetic process, from preanthesis to the postfertilization stage and in different flower organs, namely, the calyx, calyptra, anthers, filaments, ovary, and embryos. The transcript levels found were compared with the proposed model for Arabidopsis to evaluate their biological consistency. With a few exceptions, the results confirmed the expression pattern expected based on the Arabidopsis data
Identificazioni di geni associati alla tolleranza a stress osmotici attraverso il confronto dei profili trascrizionali di portainnesti resistenti e suscettibili
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