Transcriptome and proteome profiling of adventitious root development in hybrid larch (Larix kaempferi × Larix olgensis)
© Han et al.; licensee BioMed Central Ltd. 2014
Received: 12 March 2014
Accepted: 27 October 2014
Published: 26 November 2014
Hybrids of larch (Larix kaempferi × Larix olgensis) are important afforestation species in northeastern China. They are routinely propagated via rooted stem cuttings. Despite the importance of rooting, little is known about the regulation of adventitious root development in larch hybrids. 454 GS FLX Titanium technology represents a new method for characterizing the transcriptomes of non-model species. This method can be used to identify differentially expressed genes, and then two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analyses can be used to analyze their corresponding proteins. In this study, we analyzed semi-lignified cuttings of two clones of L. kaempferi × L. olgensis with different rooting capacities to study the molecular basis of adventitious root development.
We analyzed two clones; clone 25-5, with strong rooting capacity, and clone 23-12, with weak rooting capacity. We constructed four cDNA libraries from 25-5 and 23-12 at two development stages. Sequencing was conducted using the 454 pyrosequencing platform. A total of 957832 raw reads was produced; 95.07% were high-quality reads, and were assembled into 45137 contigs and 61647 singletons. The functions of the unigenes, as indicated by their Gene Ontology annotation, included diverse roles in the molecular functions, biological processes, and cellular component categories. We analyzed 75 protein spots (-fold change ≥2, P ≤0.05) by 2D-DIGE, and identified the differentially expressed proteins using MALDI-TOF/TOF MS. A joint analysis of transcriptome and proteome showed genes related to two pathways, polyamine synthesis and stress response, might play an important role on adventitious root development.
These results provide fundamental and important information for research on the molecular mechanism of adventitious root development. We also demonstrated for the first time the combined use of two important technologies as a powerful approach to advance research on non-model, but otherwise important, larch species.
Larches are very useful for afforestation because of their fast juvenile growth . Among the various larches, hybrids of Larix kaempferi × Larix olgensis are widely planted in the mountainous regions of northeastern China for their high-quality timber, their pest resistance, and their amenity value . The use of rooted stem cuttings is becoming the most popular method to propagate these hybrids. Adventitious root formation is a key step for stem cuttings, and it is a complex process affected by many factors -. Despite the ecological and economical importance of larch, little genomic research has been conducted on this species. It would be very useful to understand the biological process of adventitious root formation and development in larch.
In our previous study, two clones of L. kaempferi × L. olgensis, 25-5 and 23-12, are obviously different on the rooting rates, which were 100.0%, 94.7%, 93.3%, 96.3% and 25.1%, 19.6%, 21.5%, 22.9%, in 2001, 2002, 2003, and 2007, respectively. So, the two clones are good materials for studying the biological process of adventitious root formation. Additionally, we had examined the development process of adventitious roots in semi-lignified cuttings of hybrid larch , which consisted of three key stages: 14-18 days after cutting (DAC) for cell dedifferentiation and division; 25-35 DAC for meristem formation and development; 50-55 DAC for root formation and elongation. The early stages, 14 and 25 DAC, were crucial for the expression and regulation of rooting-related genes. These anatomical and physiological researches on adventitious roots development in hybrid larch make the further study on the molecular mechanisms governing adventitious root development feasible.
Although adventitious root formation is the key developmental process for asexual propagation of most plants, the molecular mechanisms of adventitious root formation are still poorly understood , because adventitious root formation is a complex quantitative genetic trait regulated by both environmental and endogenous factors. Only a limited number of molecular studies of adventitious root formation have been performed. Brinker et al.  identified 220 differentially expressed genes during different developmental stages of adventitious root formation in Pinus contorta hypocotyls which were treated with the auxin indole-3-butyric acid. Butler et al.  focused on gene expression patterns during adventitious root formation, and a number of mRNAs during adventitious root formation in apple were identified. Sorin et al.  identified some molecular markers which positively or negatively correlated with adventitious root formation by a proteomic analysis of different mutant genotypes of Arabidopsis. Chen et al.  provided a method suitable to study de novo root organogenesis. Ahkami et al.  identify specific genes determining the initiation and formation of adventitious roots by a microarray-based transcriptome analysis in the stem base of the cuttings of Petunia hybrida (line W115). Subramaniyam et al.  obtained a comprehensive transcript expression profiling for adventitious roots of Panax ginseng Meyer. However, very few genes were characterized for adventitious root formation of larch, due to the limited genome information.
RNA sequencing represents a powerful and rapid method for obtaining functional information on a genome-wide scale, especially for the large genomes and non-model species. Wu et al.  studied gene expression in North American ginseng root samples at seven developmental stages by 454 sequencing. Their results suggested that ginsenoside biosynthesis occurs at distinct stages of development. Firon et al.  identified the molecular mechanisms involved in the initiation of storage root formation in sweet potato by RNA sequencing. Alagna et al.  enriched the very small amount of sequence data currently available for olive, and identified genes involved in fruit quality via 454 pyrosequencing. Extensive analyses of transcriptome profiles - have identified functional genes with roles in specific biological processes. However, changes in mRNA transcript levels do not always reflect changes in protein levels, and there are many ways in which cellular events are regulated at the protein level. Therefore, a complementary protein analysis is necessary to clarify the molecular events at particular stages of development. Two-dimensional difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) has many advantages for protein analysis. Li et al.  detected significant changes in the abundance of certain proteins in cucumber roots in response to hypoxia using 2D-DIGE. Yan et al.  used a proteomic analysis to study the responses to growth restriction in maize plants supplied with sufficient nitrogen. Wang et al.  identified proteins involved in brassinosteroid regulation of rice growth using 2D-DIGE followed by MS. The results of those studies illustrated that transcriptomic or proteomic analyses are effective methods to find genes and proteins involved in regulating biological processes. Yang et al.  combined use of proteomic and transcriptomic analyses of M14 leaves and roots under NaCl treatment in sugar beet, these analyses revealed candidate genes and proteins for detailed functional characterization.
In this study, to gain a better understanding of adventitious root development in hybrid larch, we evaluated the transcriptomic profiles of two clones with different rooting capacity at two important stages of adventitious root development. Some of the genes identified as being differentially expressed between the two clones were further analyzed at the protein level. These data can remarkably expand the transcriptome and proteome database of larch, and provides a valuable platform to understand the molecular basis of regulation of adventitious root development in larch and other conifers.
Results and discussion
Functional annotation and classification
All unique sequences were first compared with sequences in the non-redundant database (nt) of the NCBI using the BLASTN algorithm. Then, they were compared with sequences in the two major public protein databases (Methods) using the BLASTX algorithm. When the E-value cutoff was set at 10-5, a total of 25965 contig sequences were annotated, which accounted for 57.5% of all contig sequences.
In principle, the higher the number of reads assembled in a contig, the higher the number of mRNA molecules encoding that gene in a given tissue sample. The read numbers of more than 700 are shown in Additional file 1. Among of the 58 contigs, 4 were involved in cell wall remodeling during plant growth and development, 10 were involved in the adaptive stress response and regulation of expression of many stress-responsive genes, and 2 were involved in polyamine synthesis. The others were involved in protein translation, synthesis, and degradation. Plant cell walls play many critical roles during plant growth, including regulation of cell differentiation, intercellular adhesion and communication, and defense against invasions by pests and pathogens -, knowing which genes are involved in the formation and remodeling of plant cell walls is of great importance. Cell wall remodeling-related genes detected in this study should be related to the wound of cuttings. Stress-related genes could enhance survival of various cell types against stress. Polyamines are key regulators in cell growth and differentiation, the detection of polyamine synthesis-related genes should be related to the different development stages of adventitious root. The detailed discussion of stress-related genes and polyamine synthesis-related genes could be found in the following sections.
Differentially expressed proteins
One notable result was that the expression patterns of S-adenosylmethionine synthetase 2 (SAMS2, spot 21, Figure 6A,B) and S-adenosyl methionine decarboxylase (SAMDC, spot 22), which are involved in polyamine synthesis, and a late-embryogenesis-abundant protein (spot 33) and an intracellular pathogenesis-related protein (spot 45), which are involved in stress responses, were the same as those detected for their encoding genes in the transcriptomic analysis. The detailed discussions of the effects of these genes on adventitious root development are showed in “elucidation of important signaling pathways” part.
Elucidation of important signaling pathways
Polyamine synthesis pathway
Genes and proteins related to polyamine synthesis and the stress response
Relative read number
S-adenosylmethionine synthase 2
S-adenosylmethionine decarboxylase proenzyme
Putative intracellular pathogenesis-related protein
S-adenosylmethionine synthetase 2
Late embryogenesis abundant protein
Intracellular pathogenesis-related protein
Stress response pathway
Cuttings excised from plants are subjected to several stresses, such as wounding and pathogen attack. The responses of cuttings to stresses are complex and involve a number of metabolic changes to protect cells and macromolecules. In this study, Contigs of UN_lyscdhit99_17285 and UN_lyscdhit99_23395 were annotated as an embryo-abundant protein and a putative intracellular pathogenesis-related protein, respectively (Table 1). Both of these proteins are related to various stress responses. We identified corresponding proteins in the proteomic profile; the embryo-abundant protein corresponded to spot 33, and the intracellular pathogenesis-related protein corresponded to spot 45.
The late embryogenesis abundant (LEA) protein family is a large family that includes proteins that accumulate at late stages of seed development or in vegetative tissues in response to some stresses. Many studies have shown that LEA proteins have important effects on salt tolerance in plants. Aghaei et al.  showed that a LEA protein was up-regulated in the hypocotyl and root of soybean treated with 100 mM NaCl. Zhang et al.  identified a putative salt-tolerance gene LEA1 in Thellungiella salsuginea, and confirmed that TsLEA1 conferred salt tolerance when expressed in yeast and transgenic Arabidopsis. Bai et al.  demonstrated that transgenic tobacco expressing the LEA3-1 protein encoded by a gene from Medicago sativa showed enhanced salt tolerance. Park et al.  showed that LEA14 might positively regulate the response to dehydration by enhancing root cell lignification in sweet potato. Overexpression of SmLEA showed faster root elongation and a higher salt and drought tolerance in Salvia miltiorrhiza . In this study, a LEA protein was annotated in the transcriptome data. The read number assembled in this gene was higher in clone 25-5 (strong rooting capacity) than in clone 23-12 (weak rooting capacity) at the root cell dedifferentiation and division stage (14 DAC). Consistent with this, the corresponding protein was expressed at higher levels in 25-5 than in 23-12 at 14 DAC (Table 1). However, the read number assembled in LEA was lower in 25-5 than in 23-12 at the root initial cell formation stage (25 DAC), and there was no significant difference in the read number assembled in LEA between 25-5 and 23-12 at 35 DAC (see Additional file 3). Together, these findings conjectured that this LEA might positively regulate the response to various stresses by enhancing lignification of cuttings, mainly at the early stage of adventitious root development.
Pathogenesis-related (PR) proteins are plant proteins induced by abiotic and biotic stresses . They accumulate around damaged cell walls to protect plants against infection by fungi, bacteria, or viruses . Many studies have shown that PR proteins have important effects on root development. Bantignies et al.  showed that a PR-10-like protein was constitutively expressed at all stages of root development in roots of white lupin. Borghi et al.  showed that PR genes affected potassium homeostasis in Arabidopsis thaliana. Takeuchi et al.  identified a root-specific PR protein induced by drought and salt treatments in rice. Immunohistochemical analyses showed that this protein strongly accumulated in cortex cells around the vascular system of roots. Koehler et al.  identified PR proteins associated with cold tolerance in strawberry. The abundance of PRprotein1.2 significantly increased in wheat seminal roots after 7 days of waterlogging . In this study, the expression of a PR protein at transcriptional and translational levels implied that it has a role in adventitious root development. It was significantly up-regulated in 25-5 (strong rooting capacity) at the adventitious root cell dedifferentiation and division stage (14 DAC) (Table 1). Therefore, it may play a crucial role in protecting the cuttings from damage caused by bacteria and fungi, mainly at the early stage after cutting, thus allowing normal adventitious root development.
In the present study, two clones of hybrid larch, different on rooting capacity, were analyzed from RNA and protein level. A transcriptome database of adventitious root development was generated included a total of 957832 raw reads, which would be useful to future molecular studies of larch. The protein profile of adventitious root development was also analyzed, 75 proteins were identified, which enriched the protein database. A joint analysis of transcriptome and proteome showed.that genes related to polyamine synthesis and the stress response might play an important role on adventitious root development. This study provides fundamental and important information for subsequent studies on the molecular mechanism of adventitious roots development. Furthermore, we demonstrated for the first time the combined use of two important technologies as a powerful approach for studying non-model, but otherwise important, larch species.
Our experimental research complied with institutional, national, or international guidelines. Field studies were conducted in accordance with local legislation, and with the permissions of forestry administrative department. We did not use any endangered species and complied with the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Voucher specimens (LH 2013001, LH 2013002, LH 2013003, LH 2013004), were identified by Prof. Xiaoshan Wang of the research institute of forestry, Bejing, and have been deposited in the herbarium of Chinese Academy of Forestry.
RNA extraction and cDNA library construction
Total RNA was prepared by the method of Sánchez et al. . The concentration and quality of total RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and checked on 1% agarose gels before proceeding (see Additional file 4). Approximately 1 μg total RNA was converted into cDNA using a SMART cDNA synthesis kit (Clontech, Palo Alto, CA, USA), optimizing the conditions to obtain a large quantity of clean cDNA in a small volume. For second-strand synthesis, PCR was carried out using a small aliquot (1/10th volume) of the primary template and Advantage 2 Polymerase Mix (Clontech). The thermal cycling program was as follows: initial denaturation at 95°C for 60 s, followed by 18 cycles of denaturation at 95°C for 15 s and annealing at 65°C for 30 s, followed by extension at 68°C for 3 min. For each sample and all PCR reactions, the PCR products were purified using a PureLink PCRTM purification kit (Invitrogen, Carlsbad, CA, USA). Double-stranded cDNA was quantified with a spectrophotometer (NanoDrop 1000, Thermo Scientific). The products were checked on a 2% agarose gel to verify cDNA quality and fragment length.
sequencing and assembly
Digested cDNA was recovered with a QIAquick PCR Purification kit (Qiagen, Hilden, Germany). Approximately 7 μg ds cDNA was sheared via nebulization into small fragments, and sequenced using the GS FLX Titanium kit. Raw unprocessed EST sequences generated from this study have been submitted to the Short Read Archive (SRA) division of Genbank. The 454 SFF file containing raw sequences and sequence quality information can be accessed through the SRA web site under the accession number SRP015266. Using the GS FLX pyrosequencing software, we selected high-quality sequences (>99.5% accuracy on single base reads) for further processing and assembly. Adapter trimming and poly (A/T) and short sequence (<50 bp) removal were performed by in-house Perl scripts to obtain clean ESTs. We used Newbler software (provided with the Roche GS FLX sequencer) for sequence assembly. The quality score threshold was set at 40.
Functional annotation and classification
The assembled unique transcripts were compared with the sequences in the non-redundant database of GenBank using the BLASTN algorithm to find and remove ribosomal RNA sequences . The remaining sequences that putatively encoded proteins were searched against the NCBI non-redundant protein (Nr) database (http://www.ncbi.nlm.nih.gov/) using the BLASTX algorithm, and against the UniProt protein database (http://www.uniprot.org/help/uniprotkb) and the KEGG protein database (http://www.genome.jp/kegg/). A typical cut-off value of E <1.0-5 was used. The GO  system was used for functional classification of sequences. GO provides a structured and controlled vocabulary to describe gene products according to three categories: molecular function, biological process, and cellular component (http://geneontology.org/).
Gene expression analysis
Normalization between the libraries
We did normalization between the libraries, including the comparison of the number of unigenes detected in each library of GO classification, and the total number of reads in each library when found the difference between two libraries. The detailed information of normalization was shown in Additional file 5.
Protein extraction and preparation
For each sample, 2 g tissue was ground into a fine powder in liquid nitrogen for protein extraction. The lyophilized powder was homogenized in lysis buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 10 mM Tris) for 30 min at room temperature with repeated shaking. The undissolved powder was removed from the homogenate by centrifugation at 40 000 × g for 60 min at 4°C. The supernatant was stored in aliquots at –80°C. Protein concentration was determined with a Bradford assay kit (BioRad, Hercules, CA, USA) using albumin diluted in lysis buffer as the internal standard.
Protein labeling with CyDye DIGE fluor
Sample lysates were labeled with Cy2, Cy3, and Cy5 following the protocols described in the Ettan DIGE User Manual (18-1164-40 Edition AA, GE Healthcare, Buckinghamshire, UK). Typically, 50 μg lysate was labeled with 400 pmol Cy3 or Cy5, while the same volume of a pooled standard containing equal quantities of all samples was labeled with Cy2. Labeling reactions were carried out in the dark on ice for 30 min before quenching with 1 mL 10 mM lysine for 10 min on ice. These labeled samples were then combined for 2D-DIGE analysis.
2D-DIGE and image analysis
We conducted 2D-DIGE as follows: The IPG strips (24 cm, pH 3–10, and NL) were rehydrated with labeled samples in the dark overnight with rehydration buffer (7 M urea, 4% w/v CHAPS, 20 mM dithiothreitol (DTT), and 1% v/v IPG buffer with a trace amount of bromophenol blue). First-dimension IEF was performed using an Ettan IPGphor System (GE Healthcare) for a total of 67 kV h at 20°C. The strips were then treated with a two-step reduction and an alkylation step prior to the second-dimension SDS-PAGE. After equilibration with a solution containing 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), 50 mM Tris-Cl, pH 8.8, and 0.5% w/v DTT, the strips were treated with the same solution except that it contained 4.5% w/v iodoacetamide instead of DTT. The strips were overlaid onto 12% polyacrylamide gels (20 × 24 cm), immobilized to a low-fluorescence glass plate, and electrophoresed for 12–18 h at 30 mA per gel using an Ettan DALT Twelve System (GE Healthcare). The Cy2, Cy3, and Cy5-labeled images were acquired using a Typhoon 9410 scanner (GE Healthcare) at excitation/emission wavelengths of 488/520 nm, 532/580 nm, and 633/670 nm, respectively. The DIGE images were analyzed by DeCyder 6.5 following the Ettan DIGE User Manual (GE Healthcare). Spot detection was performed using the DIA (differential in-gel analysis) module with the estimated number of spots set to 2500. Only spots present in all three replicate gels and qualitatively consistent in size and shape were considered. After removing artifact spots by manual editing, the DIGE images were further analyzed using the DeCyder BVA (biological variation analysis) module. A 0.05 significance level was used to define differences between groups when analyzing parallel spots. One-way ANOVA and Student-Newman-Keuls tests were conducted using the SAS software package version 8.2 (SAS Institute).
In-gel tryptic digestion and MALDI-TOF/TOF-MS analysis
Separate preparative gels were run to obtain sufficient amounts of protein for MS analysis. These gels were fixed and stained with colloidal Coomassie brilliant blue (CBB). Proteins of interest, as defined by the 2D- DIGE/DeCyder analysis, were excised from the CBB-stained gels for a modified in-gel tryptic digestion procedure. Gel pieces were first discolored in 50% acetonitrile and 25 mM ammonium bicarbonate, and then subjected to reduction and alkylation in 10 mM DTT and 55 mM iodoacetic acid, respectively. Following vacuum drying, the gel pieces were incubated with sequencing-grade modified trypsin (Promega, Madison, WI, USA) at a final concentration of 0.01 mg/mL in 25 mM ammonium bicarbonate for 16 h at 37°C. Supernatants were collected, vacuum dried, and then redissolved in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA) for MS analysis using an ABI 4800 Proteomics Analyzer MALDI-TOF/TOF-MS. The TOF spectra were recorded in the positive ion reflector mode with a mass range from 700 to 4000 Da, and 10 of the strongest peaks in each sample were chosen for MS/MS analysis. The spectra were corrected by an external standard method using trypsin-treated myoglobin peptides. The MS/MS results were searched using GPS (Applied Biosystems, Foster City, CA, USA) - MASCOT (Matrix Science, London, UK) with the following criteria: NCBInr database; species restriction, green plants; MS tolerance was set at 6100 ppm and MS/MS at 60.6 Da; at most one missed cleavage site; fixed modification was carbamidomethyl (Cys) and variable modification was oxidation (Met); and cleavage by trypsin was on the C-terminal side of Lys and Arg unless the next residue was Pro. If peptides matched to multiple members of a protein family, or if a protein appeared under different names and accession numbers, the entry with the highest score was selected. In addition, the theoretical molecular weights and pI of the identified proteins were calculated using the Peptide Mass program (http://web.expasy.org/peptide_mass/).
Availability of supporting data
The raw sequences data supporting the results of this article are available in the Short Read Archive (SRA) (accession number SRP015266), http://www.ncbi.nlm.nih.gov/sra.
This study was financially supported by the National Science and Technology Support Program (2012BAD01B01) and the National Science Foundation (30972393). The authors thank Dr. Liang Zhang for the service of cDNA library construction and 454 sequencing. The authors are also grateful to Edanz (http://www.liwenbianji.cn/) for their helpful suggestions on improving the manuscript.
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