Genetica DOI 10.1007/s10709-013-9743-4
Comparative transcriptional analysis reveals differential gene expression between Sand Daffodil tissues Bruna De Felice • Francesco Manfellotto • Raffaella D’Alessandro • Olga De Castro • Antonietta Di Maio • Marco Trifuoggi
Received: 4 January 2013 / Accepted: 14 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Sand Daffodil (Pancratium maritimum) is a world-wide endangered Amayllidaceae species and represents an important anti-cancer medicinal resource due to alkaloids production. Despite its increasing pharmaceutical importance, there are not molecular resources that can be utilized toward improving genetic traits. In our research, the suppression subtractive hybridization (SSH) method conducted to generate large-scale expressed sequence tags (EST), was designed to identify gene candidates related to the morphological and physiological differences between the two tissues, leaves and bulbs, since lycorine, the main anti-cancer compound, is there synthesized. We focused on identification of transcripts in different tissues from Sand Daffodil using PCR-based suppression SSH to identify genes involved in global pathway control. Sequencing of 2,000 differentially screened clones from the SSH libraries resulted in 136 unigenes. Functional annotation and gene ontology analysis of up-regulated EST libraries showed several known biosynthetic genes and novel transcripts that may be involved in signaling, cellular transport, or metabolism. Real time RT-PCR analysis of a set of 8 candidate genes further confirmed the differential gene expression. B. De Felice (&) F. Manfellotto R. D’Alessandro DISTABIF-Department of Science and Technology, Environmental, Biological and Pharmaceutical, University of Naples II, Via Vivaldi 43, 81100 Caserta, Italy e-mail:
[email protected] O. De Castro A. Di Maio Department of Biological Sciences, Section Plant Biology, University of Naples Federico II, Via Foria, Naples, Italy M. Trifuoggi Department of Chemistry, University of Naples Federico II, Naples, Italy
Keywords Suppression subtractive hybridization (SSH) Sand Daffodil Lycorine Anti-cancer compounds
Introduction The genus Pancratium of Amayllidaceae comprises approximately 15 species, which are distributed along the Mediterranean coastal area and extending to the Canary Islands, tropical Africa and tropical Asia. The objective of this study was to identify tissue-specific genes in Pancratium maritimum, or sea daffodil, a species of Amaryllidaceae native to the Mediterranean region and south-western Europe. It grows on coastal sands or just above the high tide mark. Other vernacular names are Sand Daffodil, Sand Lily and the Lily of St. Nicholas. The Latin maritimum means ‘‘of the seashore’’. P. maritimum is a bulbous perennial with a long neck and glaucous, broadly linear leaves, evergreen, but the leaves often die back during hot summers. In consequence of the lost of its natural habitats and populations the species is endangered along Mediterranean coasts (De Castro et al. 2012; Di Maio and De Castro 2013a , b; Zahreddine et al. 2004) and under protection by the Biodiversity Act (2002). Profound interest have aroused several compounds with pharmacological and apoptotic in cancer cell lines activity found in P. maritimum making it a suitable anti-cancer agent (Lamoral-Theys et al. 2009). One of these is lycorine, mainly expressed in bulbs and leaves (Sanaa et al. 2010). Harvest from natural sources could be often insufficient to provide adequate quantities of the product of interest in a sustainable manner. The development of in vitro techniques for screening and producing of these alkaloids could be an important technological step by the
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mean to prevent the natural populations lost as well as to ensure the continuous supply of these substances to pharmacy. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving plant traits and the molecular genetics of alkaloids biosynthesis is not fully elucidated. Despite the development of genomic resources, the genome of Pancratium maritimum is still unknown, and, at the present, the knowledge of P. maritimum genome and transcriptome is currently restricted to the 43 sequences available in Gen-Bank (Nucleotide, September 2011). The aim of our research was to analyzed transcriptome features of P. maritimum in different tissue (bulbs and leaves) to improve our understanding about the molecular mechanisms of tissue specificity in P. maritimum and to clarify the overall characteristics of expression profiles associated with development and the molecular programs. Beside, this study could be useful in expanding our understanding of the molecular genetics of the complex lycorine biosynthesis process, which is mainly expressed in bulbs and leaves. Using a PCR-based suppression subtractive hybridization strategy, ESTs were isolated from leaves and bulb Sand Daffodil libraries. Expression analysis of randomly selected differential-regulated ESTs from the SSH libraries was also performed using macro arrays. Computational functional annotation and gene ontology (GO) analysis of up-regulated EST libraries showed several homologous genes as well as novel transcripts. Real Time RT-PCR analysis of a set of 10 candidate genes further confirmed the gene expression in the two libraries. Interestingly, among other genes, several up-regulated genes that are potential candidates for regulation of drug transportation and accumulation as lycorine were obtained from libraries screening. Beside, several long terminal repeat (LTR) retrotransposons-like sequences have been found transcriptionally active in bulbs and leaves. We showed that the fraction of LTR retrotransposons -related transcripts varies greatly among LTR elements classes and among tissues. Our results provide insights into the genes and molecular mechanisms underlying the development and tissues specificity in P. maritimum, characterizing the key components of the regulatory network regulating the processes.
Materials and methods Plant material and RNA isolation Plant material was collected from species from the P. maritimum. The plants were from the collection of the Botanical Garden of Naples, Italy.
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DNA was extracted from the young leaves using DNeasy Plant Mini Kit (Quiagen Inc., Mississauga, CA). RNA was extracted using Trizol method and total RNA was extracted according to the manufacturer’s protocol (Invitrogen Corporation, Carlsbad,CA). A DNAse I treatment was applied to remove traces of contaminating DNA. The total RNA was purified using the RNeasy Mini Kit (QIAGEN Inc., Mississauga, CA). The RNA concentrations were determined; the A260/A280 and A260/A230 ratios were calculated as indices of protein and volatile compound contamination, respectively, using a spectrophotometer (NanoDropND-1000;Nano Drop Technologies, Inc.,Wilmington, DE, USA). The integrity of the total RNA was determined by electrophoresis on a 1.5 % denatured agarose formaldehyde gel stained with ethidium bromide. Suppression subtractive hybridization (SSH) libraries Suppression Subtractive Hybridization libraries using leaves and bulbs from ten plants of P. maritimum were generated using the Clontech PCR-Select cDNA Subtraction Kit (Takara Bio Inc., Clontech, Mountain View, CA) according to the manufacturer’s instructions. For each SSH library two different double stranded cDNA pools (a ‘tester’ and ‘driver’) were used. Library products were separated on agarose gels. For each library, products below and above 500 base pairs were purified separately using Gel Purification Kit (Qiagen, Valencia, CA) and cloned into TOPO 2-1 vector (Invitrogen, Carlsbad, CA). Expression analysis by macroarray Macro arrays constructed for this study comprised 1,000 SSH fragments from both the leaves and bulbs libraries. Bacterial cultures were grown overnight in LB media containing 50 lg/mL kanamycin. Plasmid DNA was purified using QIAprep-miniprep (Qiagen, Valencia, CA). SSH fragments were amplified by PCR using adapter specific primers. Amplification of fragments via PCR was performed using ExTaq Hot Start Polymerase System (Takara Mirus Bio, Madison, WI). PCR conditions were as follows: 95 °C for 2 min followed by 259: 30 s, 95 °C; 30 s, 62 °C; 2 min, 72 °C. This was then followed by 19: 5 min, 72 °C. PCR products were precipitated, dissolved in water and denatured in 0.01 % sodium dodecyl sulfate (SDS), 0.2 N NaOH at 72 °C for 15 min. Denatured PCR products were printed in duplicate on Zetaprobe membranes (BioRad, Hercules, CA) using hand held 96-pin replicating tool (V&P Scientific, San Diego, CA). Positive control cDNA (5.8 s rRNA) was also transferred on each membrane and as negative control 5 lg of pCR2.1TOPO TA vector was spotted. For each SSH-
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subtracted library plates, three identical cDNA macro array membranes were generated. Before use as probes, tester and driver pools were normalised by one round of subtraction in which the tester probe was subtracted with the driver and driver with tester using the PCR-select cDNA subtraction kit (Clontech). These cDNA probes were labeled with [a-32P] dCTP using the Rediprime II random primer labeling system (Amersham Biosciences). Fifty nanogram of the same SSH subtractive cDNAs were marked as probe labeled by random priming with a32P[dCTP] to hybrid with the three repeated copies membranes of the same cDNA library. SSH-macroarray membrane replicates were hybridized with identical amounts (cpm) of specific heat denatured cDNA probe (SH-leaves, SH-bulbs). Macro array blots were submerged in neutralization solution (0.5 M Tris pH 7.4, 1.5 M NaCl), then in 69 sodium citrate/sodium phosphate (SCP) for 1 min prior to UV cross-linking. Pre-hybridization of the membranes occurred for 8 h in 10 % dextran sulfate (average molecular weight of 500,000 kD; G.E. Health, Piscataway, NJ), 5.5X SCP, 0.9 % n-lauroyl sulfate-sodium salt and 0.5 mg/ ml heparin. Hybridization was performed at 65 °C for 18 H, followed by washing twice with 2X SCP, 0.1 % SDS for 15 min then twice using 0.29 SCP and 0.2 % SDS for 15 min at 65 °C. Blots were exposed to phosphorimager screen for 24–48 H then read by FLA-5000 phosphorimager (Fujifilm Medical Systems Inc., Stamford, CT). Probes were stripped off of blots up to three times using 0.1 % SDS at 100 °C. Blots were checked for residual hybridization by exposing to film for 24 H before re-hybridizing with a different target cDNA. Signal intensities of each spot were captured using VisualGrid (GPC Biotech Inc, Waltham, MA). Following local background subtraction, the intensity values for each spot were globally normalized using LOWESS (locally weighted polynomial regression) within and across all three technical replicate experiments. The relative fold change expression signal obtained from each clone found significant in one tailed student t test were considered for further analysis (C2 fold for up and down).Additionally, relative fold change of three or more independent ESTs corresponding to a particular contig were analysed using Q-test with 95 % confidence for finding outlying data points. Gene identities and bioinformatics Plasmids from independent clones were isolated and were sequenced from both strands using the Big Dye Terminator Cycle Sequencing Kit via the automatic sequencing system ABI PRISM 310 DNA Sequencer (Applied Biosystems).
All EST sequences were screened through VecScreen software (NCBI:http://www.ncbi.nlm.nih.gov) to remove vector sequence contamination. Once cleaned of vector sequence, all the EST sequences from each up-regulated library and from each down regulated library were used separately for contig assembly. The ESTs were grouped into contigs and singletons and were termed as unigenes. The unigene sequences were used to search for homology using the default setting of BLASTX program at NCBI. Each unigene was classified as a protein with known function, a protein with unknown function or a protein with no sequence match in the database. Furthermore, sequences with BLASTx hits were annotated according to GO terms using Blast2GO software (www.blast2go.org). The TrEMBL section of UniProtKB (http://www.uniprot.org) database was used to perform, enzyme class and pathway analysis. Validation step: Gene expression analysis by Real-time qRT-PCR Primer pairs were designed for each sequence using Primer Express software v3.0 (Applied Biosystems) and following the manufacturer’s guidelines for primer design. For SYBR Green real-time RT-PCR assays, the amplification efficiency of all primer pairs was optimized with genomic DNA from P. maritimum bulbs and leaves using the StepOnePlus Real-Time PCR System (Applied Biosystems). RT-PCR was performed using the total RNA used to make the SSH libraries. Total RNA (500 ng in a final volume of 20 lL) was reverse-transcribed using the PrimeScript II 1st strand cDNA synthesis kit (TaKaRa) according to the manufacturer’s protocol. Real-time quantitative PCR was performed using the Power SYBR. Green PCR Master Mix (Applied Biosystems) on the StepOnePlus Real-Time PCR System (Applied Biosystems). PCR mixtures were prepared according to the manufacturer’s instructions and contained 300 nM of both the forward and reverse gene-specific primers and 4 lL of the 20-fold diluted reverse transcription reaction (total 5 ng) in a final volume of 20 lL. All reactions were heated to 95 °C for 10 min; this denaturation step was followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The PCR products were subjected to melting curve analysis; the conditions were incubation at 60–95 °C with a temperature increment of 0.3 °C s–1. 5.8 s rRNA was used as the reference gene for normalizing the transcript profiles. DDCt method was used to quantify relative transcript abundance (Livak and Schmittgenn 2001). All assays were carried out in triplicate, and the data are presented as mean ± SD of three replicates. The specificity of each amplification was checked by melting analysis and agarose gel electrophoresis of the amplified products.
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Isolation of retrotransposon sequences by PCR
Statistical analysis
Sequences of transposable TY copia-like LTR Reverse Transcriptase were obtained using the primers Ty1-R (50 ARCATRTCRTCNACRTA) and Ty1-F (50 ACNG CNTTYYTNCAYGG), as described by Flavell et al. (Flavell et al. 1992). Sequence accessions were aligned and conservation assessed with the multiple alignment procedure of ClustalW (Thompson et al. 1994). Degenerate primers were developed with the program FastPCR and were based on most conserved aligned Ty1-copia -like reverse transcriptase (rt) amino acid sequences from plants. We designed several primer pairs for the RT element to compare the efficiency and reproducibility of amplification. None of the primer pairs that were chosen formed dimers and all show high PCR efficiency. PCR was performed on an a Techne (Staffordshire, UK) PTC-100 thermal cycler under the following conditions: 3 min at 94 °C, followed by 31 cycles of 30 s at 94 °C, 30 s at 45 °C, and 60 s at 72 °C, and a final extension of 3 min at 72 °C. Individual PCR reactions each contained 5 ng DNA, 50 pmol of each primer, 1 unit of Taq polymerase, and a final concentration of 50 mM KCl, 2 mM MgCl2, and 100 lM of each dinucleotide triphosphate. In our analysis of retrotransposon elements in P.maritimum, we obtained conspicuous DNA bands of about 300 bp. It was electrophoresed on 1.5 % agarose gels containing ethidium bromide, cut from the gel, purified with the Qiaquick Gel Extraction Kit (Qiagen), cloned into the pMOSBlue vector (pMOSBlue blunt ended cloning kit, Amersham Pharmacia Biotech), and used to transform the DH5a competent cells (Stratagene).
Data are presented as mean ± SD. Differences between groups were compared at each time point using Wilcoxon tests and differences within groups were evaluated using Mann–Whitney test. Bilateral tests were used for all analyses. P \ 0.05 was considered significant. GenBank Accession Numbers Ty1-copia sequences were submitted to the Gen-Bank databases. The assigned GenBank Accession numbers JX391843-45 and JN828812.
Results Differential screening of ESTs Pancratium maritimum forms an important anti-cancer medicinal resource, producing compounds as lycorine, mainly in bulbs and leaves (Sanaa et al. 2010). These tissues were used to detect differentially expressed transcripts with a PCR-based cDNA subtraction method. A distinct driver/tester combination was used to construct two different SSH libraries. The combinations of driver/tester were as follows: driver = bulbs; tester = leaves used to construct the forward and Reverse Subtracted cDNA library which contained 1,000 clones each by SSH.
Sequences of TE elements and phylogenetic analysis
Evaluation of tissue differential expression using macroarrays
Recombinant clones from TE elements cloning were sequenced from both strands using the Big Dye Terminator Cycle Sequencing Kit via the automatic sequencing system ABI PRISM 310 DNA Sequencer (Applied Biosystems). The cloned sequences were analyzed for homology to known sequences to DNA (BLASTN) and proteins (BLASTX) using the NCBI. The nucleotide sequences were translated for all six possible reading frames, using the ExPASy translation tool. The closest matches to known sequences from the NCBI blast searches were retrieved and aligned to those sequenced using the Clustal W program (Thompson et al. 1994). A phylogenetic tree was constructed from the evolutionary distance matrix based on the Kimura two parameter algorithm using the neighbourjoining method (Saitou et al. Saitou and Nei 1987). The analysis was performed with the Mega4 program (Tamura et al. 2007), and gap sites in the alignment were excluded in the pairwise comparison.
Differential hybridization screening was performed using macroarrays in order to isolate genes differentially expressed in leaves and bulb tissues from the 1,000 randomly selected clones. Differentially expressed cDNA clones were identified based on the hybridization signal intensities observed between the 2 membranes. Representative differential screening results are illustrated in Fig. 1. Of the cDNA clones screened, 294 were identified as strongly expressed and were analyzed on agarose gel. Sequencing performed on single band PCR amplicons generated a total of 224 clones with adequate sequencing results for BLAST analysis. 224 positive clones of strongest signals were sequenced so that 119 EST sequences of leaves and 105 ESTs of bulbs were obtained. The EST sequences of corresponding clones were trimmed by removing the vector sequences and fuzzy sequences from the former sequences of bases and got 200 in all.
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Fig. 1 Differential hybridization screening. Representative differential screening results of macroarrays of the SSH-libraries. SSH-leaves subtracted cDNAs probe (a), SSH-bulbs subtracted cDNAs probes (b), C- indicates pCR2.1 TOPO TA vector; C?, 0.5 lg of cDNA (5.8 s rRNA). The arrow indicates an example of differentially expressed genes in SSH-leaves compared to SSH-bulbs
Bioinformatics analysis The cleaned unigenes/non-redundant sequences were assembled with more than 20,000 EST sequences deposited in GenBank. Database searches and similarity analyses of cDNA nucleotide sequences were carried out with the BLASTN and BLASTX programs against public nucleotide, EST, and protein databases. Total 137 unigenes with 101 from leaves and 36 from bulbs were obtained (Supplementary Tab.1). Functional classification of the unigenes Putative functional annotation of the unigenes was assigned by BLASTX analysis against the GenBank nonredundant database for sequence similarity (E \ 10-5). Unigenes showing BLASTX homology at E \ 10-5 were designated as unigenes with no match in the database. Approximately 46 % of the unigenes fell into this category, and may be specific to P. maritimum species. Approximately 12 % of the sequences represented evolutionarily conserved proteins whose function is not known. Several novel transcripts were identified. Functional classification of P. maritimum up-regulated unigenes, from leaves and from bulbs showing sequence similarities to known or putative function proteins, was determined based on GO analysis for biological process, molecular function and cellular components (Fig. 2). In the cellular component category, proteins associated with intracellular organelles, membranes and cytoplasmic parts were most enriched.
The GO annotation showed the cellular component of bulbs were mainly located in cytoplasm and nucleus, reaching to 58 %; while leaves also mainly located in cytoplasm and nucleus, especially located in plasmodesm, integral to membrane and endoplasmic reticulum (Fig. 2a). The main biological functions of leaves were related to DNA repair/replication, nucleic acid binding, ATP and ion binding, RNA-directed DNA polymerase activity, oxidoreductase activity. The bulbs expressed more transcripts especially in signal transduction, as kinase and transferase activity, transport, calcium binding, peptidase and helicase activity (Fig. 2b). According to the information of GO annotation, the leaves tend to growth and self-protection, while the bulbs tend to more active in metabolism. Polysaccharide and metal ion binding genes were expressed on both leaves and bulbs. Eighteen genes have not been annotated. A group of ESTs contained Ty1-copia retrotransposons sequences. This is of relevance, since even if transposable elements (TEs) transcription activity has been only demonstrated for a few plants and only activated under stresses conditions (Grandbastien 1998; De Felice et al. 2009), in P. maritimum despite mutations and cell control, Ty1-copia sequences manage to be transcriptionally active in leaves and bulbs. These annotations provide a valuable resource for investigating specific processes, functions and pathways and allow for the identification of novel genes involved in the pathways of secondary metabolite synthesis. Validation of differential expression using selected SSH clones and quantitative real-time PCR (qRT-PCR). To validate the results of the SSH procedures, expression of 8 ESTs recovered in both of the two libraries was assayed using qRT-PCR; samples for the qRT-PCR analysis were collected from P.maritimum leaves and bulbs. The results of the qRT-PCR analysis and SSH were consistent (Fig. 3). Based on qRT-PCR analysis, six ESTs-putatively encoding major facilitator superfamily (MFS), MFP and RND transporter, putative receptor serine/threonine kinase PR5 K, delta-9 desaturase-like 5, esterase/lipase/thioesterase protein, were upregulated in leaves, while putative puroindoline b protein and DEAH box helicase were downregulated compared to bulbs. Isolation of novel retrotransposon sequences Since we found that retrotransposons were differentially expressed during P. maritimum development, we have also scanned its genome using PCR targeting of the LTR reverse transcriptase domains of retrotransposons. The multiple alignments showed that we had isolated 18 novel
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Fig. 2 Gene ontology mapping of differentially expressed genes from bulbs and leaves SSH libraries in Sand Daffodil using BLAST2GO automated sequence annotation. Sequences from our libraries displaying BLASTx 1 9 values \ 19E-05 underwent GO assignment. EST distribution in the categories a cellular components
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from bulbs and leaves SSH libraries; b cellular components from bulbs SSH library; c cellular components from leaves SSH library. d Molecular function from bulbs and leaves SSH libraries; e molecular function from bulbs SSH library; f molecular function from leaves SSH library
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Fig. 3 Quantitative real-time PCR of transcripts differentially expressed in Sand Daffodil bulbs and leaves. 5.8 s rRNA was used as the reference gene, and the data were calibrated relative to the transcript levels in Sand Daffodil bulbs and leaves. The data are presented as the mean ± SD of three replicates. P \ 0.05 was considered significant
types of Ty1-copia-like LTR reverse transcriptase from P. maritimum (Fig. 4).
Discussion Suppression subtractive hybridization is a powerful genomics technique that enriches differentially regulated genes and has mostly been used to identify transcripts expressed in contrasting conditions. SSH is a suitable approach in P. maritimum where the genomic information is currently scarce. Studies on P. maritimum genetic expression have not been carried out and research of its tissue-specific transcriptome is absent. There is also no study concerning the association between the plant transcriptome and metabolome with respect to the plant tissue type. Therefore, we used the PCR-based SSH approach to identify transcriptional differences between leaves and bulbs from P. maritimum, where lycorine is mainly expressed. Gene expression profiling is an important strategy for obtaining knowledge on presumed function of genes that comprise animals and plants (Schmid et al. 2005). Subsequently, complete annotation of every transcriptional unit has become an enormous challenge not only for a complete understanding of the biology of plants, but more importantly, for efficient utilization of that information for genomics-based crop improvement (Ohyanagi et al. 2006).
Analyses of P. maritimum transcriptome encompassing different cell types, tissues and organs, specific stages of growth and development, and specific treatment conditions could generate a large amount of information that provides initial clues for understanding the function of genes based on their time, place and level of expression in the plant. In order to understand these transcriptional programs reflecting physiological and alkaloids production states it is essential to monitor the expression profiles of a large number of genes, including uncharacterized ones, throughout the life cycle of P. maritimum in the field. With respect to gene expression, transcriptomes from different plants or organs at different physiological states can be monitored and compared (Seki et al. 2004). Moreover, large-scale single-pass sequencing of cDNA prepared from tissues with specific metabolism activity has provided an efficient and rapid mean towards isolation of new genes (Suh et al. 2003). It has been reported that although the synthesis of alkaloids as lycorine occurs in the leaves, it is stored in the bulbs (Evidente et al. 1985). Up-regulated genes that are potential candidates for regulation of drug transportation and accumulation were obtained from library screening. One such gene encodes the MFS transporters make use of proton gradient across the plasma membrane for the extrusion of drugs. It consists of evolutionary conserved membrane transport proteins involved in the symport, antiport or uniport of various substrates (De Rossi et al. 2002). MFS proteins also function as major drug transporters, which are involved in drug efflux. Membrane Fusion Proteins (MFPs) are functional subunits of multicomponent transporters that perform diverse physiological functions in both Gram-positive and Gram-negative bacteria functioning in multidrug efflux. The MFP-dependent transport seems to have advantage when substrates must be prevented from crossing the cytoplasmic membrane or when the exported endogenous substrate is highly toxic and must be secreted with the least number of intermediate steps. The largest and the best characterized group of MFPdependent transporters are those involved in multidrug efflux in Gram-negative bacteria (Lomovskaya et al. 2008). In association with the RND- or the MF-type inner membrane transporter and the OMF channel, these MFPs enable highly effective extrusion of multiple structurally unrelated compounds. They are able to pump out literally thousands of antimicrobial agents, solvents and even hormones, Among known metabolites exported by MFP-dependent transporters are indoles, siderophores, porphyrin(ogen)s, quorum sensing signals, pigments, electron shuttles and oligosaccharides (Zgurskaya et al. 2009). A secretion protein, RND family efflux encoding gene, represents a small family of drug and heavy metal efflux permeases, now commonly referred to as the RND family.
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Genetica Fig. 4 Phylogenetic analysis of translated amino acid sequences representing the Ty1-copia-like RT PCR fragments isolated from Sand Daffodil. Numerals at the branch nodes indicate the bootstrap support out of 1,000 replications. The branch lengths are proportional to the genetic distances as estimated by the neighbor-joining method
This family is actually a ubiquitous superfamily with representation in all major kingdoms (Tseng et al. 1999). Further experiments will allow to understand if the regulation of such genes is related to alkaloids and lycorine accumulation in Sand Daffodil tissues. The library was also enriched with up-regulated genes that presumably have no direct role in alkaloids accumulation. Good examples are the putative Tir-nbs-lrr resistance protein, pathogen resistance gene, Desaturases, that introduce double bonds into the fatty acids are involved in the adaptation of membrane fluidity to changes in the environment, all of which are abiotic and biotic resistance-related genes that have well-characterized roles in model plant species (Tseng et al. 1999; Gostincar et al. 2010). For several transcripts, a reliable prediction of general biochemical activity can be made, but predictions neither suggest nor challenge a role in alkaloids synthesis. Genes in this class include three receptor serine/threonine kinase, which show
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significant similarity to the several known plant serine/ threonine kinase, and several nucleic acid binding protein encoding genes. Any of these genes could participate in the uncharacterized steps of the biosynthetic pathway but could equally well be unrelated to lycorine synthesis. Further characterization will be necessary to determine whether these genes have direct relevance to alkaloids biosynthesis. Genes orthologous to the RNA helicases of the DEAHbox protein family were found in P. maritimum genome. The expression profile showed that DEAH helicases were down-regulated in leaves compared to bulbs (Fig. 3), suggesting that these genes have similar functions in bulb and leaf. The DEAH/DEAD box helicases are a family of proteins whose purpose is to unwind nucleic acids. They are involved in various aspects of RNA metabolism such as nuclear transcription, pre mRNA splicing, ribosome biogenesis, nucleocytoplasmic transport, translation, RNA decay and organellar gene expression. Helicases might be
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playing an important role in regulating plant growth and development under stress conditions by regulating some stress-induced pathways. There are now few reports on the up-regulation of DEAH-box helicases in response to abiotic stresses. Recently, salinity-stress tolerant tobacco plants have already been raised by overexpressing a helicase gene (Vashish and Tuteja 2006) Recent reports (Macovei and Tuteja 2012) for developing salinity tolerant plant by overexpressing a helicase gene (PDH45) speculate that the mechanism by which the helicase gene confers stress tolerance, likely involves regulation of translation initiation, a known function of eIF-4A, or of transcription regulation, through its RNA helicase function. Recent reports indicate that the expression of genes encoding helicases is also regulated in response to changes in specific environmental conditions, including temperature, light, oxygen or osmolarity (Owttrim 2006). For example, the ectopical expression of PDH45 (Pea DNA Helicase 45) helicase, a member of the DEAD-box protein family, resulted into salinity stress tolerance both in tobacco and rice plants Mishra et al. 2005). Based on sequence and expression data analogies with corresponding Triticum gene data, two Puroindoline b-related sequences were isolated in P. maritimum. Both of the P. maritimum genes were over-expressed in the bulbs compared to leaves (Fig. 3). Puroindoline, endosperm-specific proteins involved in Triticum wheat seed hardness, are small proteins reported to have in vitro antimicrobial properties (Dixon and Harrison 1990). Gautier et al. (2000) suggest that the difference in tolerance of common and durum wheat to disease, especially Fusarium head blight, could be related to their Pin genotypes, durum wheat lacking these genes. Synthetic peptides of PINA and PINB exhibit significant antimicrobial activity against both Gram-positive and Gram-negative bacteria (Jing et al. 2003), and their potential as antimicrobial agents is further supported by studies using model membranes and/or microbial and fungal cells (Rezansoff et al. 2005; Vila-Perello et al. 2006). This is noteworthy, since it can be important to express PIN proteins in other plants to test their antimicrobial potential and any differences therein (Bhave and Morris 2008). Plastic genes as ribosomal proteins and many other DNA/ RNA metabolic process-related genes (Fig. 2) were identified in both libraries, which are worthy of further investigation to determine their specific impact on Sand Daffodil development parameters. In fact, the modulation of ribosomal proteins suggests that transcriptome reprogramming during development involves a shift in protein synthesis. Beside, the modulation of ribosomal proteins in plants has been reported in response to various forms of abiotic stress including dryness (McHale et al. 2006), and salinity (Mukhopadhyay et al. 2011). Other genes in the DNA/RNA metabolic process category were related to stress responses and recovery, which could affects the ability of Sand Daffodil to adapt to dry and saline coastal sands.
A class of genes (29 %) has functions that cannot be predicted based on sequence similarity information. These genes may be involved in important but uncharacterized processes related to alkaloids accumulation. As many as 8 % of these ESTs showing no sequence homology with any of the available Genbank sequences may be specific to Pancratium species. The other 11 % of sequences are similar to genes described as encoding hypothetical proteins or proteins with unknown function. Sequencing of the total Pancratium transcriptome will be required to identify transcripts for every gene to find out if they are related to alkaloids accumulation. Interestingly, the library was also enriched with a group of LTR-retrotransposable elements having a considerable transcriptional activity in P. maritimum leaves and bulbs, suggesting that, either transposition are more frequent than previously expected, or cells can control transposition at a post-transcriptional level. However, although epigenetic changes may explain activation of certain retrotransposons in some tissues, not all retrotransposon families accumulate equally, suggesting that such occurrence requires some family specific mechanisms, rather than being the result of a genome-wide activation of retrotransposons. One possible explanation may be the presence of cis specific signals in the retrotransposons promoter that may enhance their expression in certain cells. Transposable elements transcription has been believed to be severely repressed in plants. This point of view was supported by the fact that during long time transcription activity was only demonstrated for a few plant TEs, and only activated under certain precise circumstances as, for example, pathogen infection, physical injuries or different abiotic stresses (Grandbastien 1998; De Felice et al. 2009). Our data indicate that transposase-like genes can be essential for plant development and can also regulate global gene expression. Thus, transposases can become domesticated by the host to accomplish significant cellular functions. Transposon sequences apparently have the potential to evolve functions that are essential for plant growth and development. Given the large number of retrotransposon copies that we identified in P. maritimum genome and the fact that retrotransposons managed to be transcriptionally active, further studies on retrotransposon transcription and their consequences in P. maritimum will enable us to understand the role of transposable elements in this medicinal plant.
Conclusions At our knowledge, this is the first study reporting the identification and the characterization of differentially expressed sequences in P. maritimum. The analysis of every transcriptional unit and the genomic distribution
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