- Research article
- Open Access
Genome-wide transcriptional analysis of super-embryogenic Medicago truncatulaexplant cultures
© Imin et al; licensee BioMed Central Ltd. 2008
- Received: 22 May 2008
- Accepted: 27 October 2008
- Published: 27 October 2008
The Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip®, we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation.
Using Affymetrix GeneChip®, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower promoting factor 1, nodulin 3, fasciclin and lipoxygenase, and a down-regulated gene ETHYLENE INSENSITIVE 3, all of which positively correlated with the microarray data.
We have described the differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong at an early time point. This data will facilitate the mapping of regulatory and metabolic networks involved in the gaining totipotency and regeneration capacity in M. truncatula and provides candidate genes for functional analysis.
- Somatic Embryogenesis
- Embryogenic Culture
- Somatic Embryo Formation
- Phytohormone Biosynthesis
- Medicago GeneChip
Plants are well known for their extraordinary capacity to regenerate whole organisms from somatic cells. They often retain plasticity and have the capability to reverse the differentiation process and change their fate. The remarkable plasticity of plant cells is well exemplified by the capability of differentiated leaf cells to retain totipotency, the ability of a single cell to develop into a new organism . This process is known as somatic or asexual embryogenesis (SE) whereby somatic cells differentiate into embryos and ultimately into plants via a series of characteristic morphological stages, particularly the later stages, which resemble the zygotic stages of development [2, 3]. SE is the developmental restructuring of somatic cells towards the embryogenic pathway and forms the basis of cellular totipotency in higher plants [4, 5]. Analyses of gene expression during somatic embryogenesis can provide information about the early stages of plant development . Large-scale transcription analyses of embryogenesis have also been reported in several species [6–12]. Numerous genes have been identified as specifically expressed during somatic embryogenesis [13, 14]. These genes include hormone responsive genes such as auxin inducible genes , late embryo abundant genes , calmodulin , calcium dependent/calmodulin-independent protein kinases , calmodulin-like protein kinases , somatic embryogenesis receptor-like kinase (SERK) genes [3, 4, 20], homeobox containing genes [21, 22]; chitinases ; arabinogalactans , lipid transfer proteins , WUSCHEL  and LEAFY COTYLEDON genes [27, 28], to name a few. As yet little is known about the induction and maintenance process of the genes involved in the SE processes, especially in the acquisition of totipotency of somatic cells. Avivi et al. has shown that that acquisition of pluripotentiality involves changes in DNA methylation pattern and reorganisation of specific chromosomal subdomains. These changes lead to activation of silent genes such as plant specific NAC (no apical meristem-like) genes and VIP1, a gene encoding b-Zip nuclear protein that involved in acquisition or maintenance of pluripotentiality . Several researchers have sought to identify the very early plant cells in the explant cell population that are competent to be committed to differentiation pathways. Using the SERK gene as a marker during the examination of either carrot hypocotyls explants , immature zygotic embryos of sunflower , leaf explants of Dactylis glomerata L. (Poaceae) , or developing ovules and embryos of Arabidopsis . SERK gene is expressed early in a small sub-population of cells which are competent to form embryogenic cells . Over-expression of the AtSERK1 gene in Arabidopsis cultures was shown to induce somatic embryo formation . Similarly, the over-expression of a transcription factor called BABY BOOM (BBM) that shows similarity to the AP2/EREPB multigene family of transcription factors  under the control of the 35S promoter in transgenic plants induced ectopic spontaneous somatic embryos and cotyledon-like structures on Arabidopsis and Brassica seedlings. The BBM gene was originally isolated because it represented a gene that was expressed early in the initiation of the differentiation of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis) and appeared to be involved in the conversion from vegetative to embryonic growth .
Legumes in general have proven recalcitrant at de novo regeneration in vitro . In Medicago truncatula, leaf explants as well as protoplasts can form calli and subsequently the generation of embryos and then the development of plants . Depending on the plant system, auxin and/or cytokinin are required to enable embryogenesis to occur in culture [3, 30, 31, 34]. In Medicago truncatula, Nolan et al. found that embryogenesis required both auxin and cytokinin addition, although some embryos could form on cytokinin alone . In the leaf explant tissue culture system, there is an advantage of being able to manipulate the type of differentiating cells observed by changing the phytohormones added to the culturing media [3, 4, 20], and embryos are initiated more rapidly in 4–6 weeks. This meristematic system has ideal attributes: the regenerative capacity of the mutant line 2HA, which is 500 fold more embryogenic than its isogenic line Jemalong [34, 35]. When both M. truncatula cultivar (cv) Jemalong and 2HA explant tissues are cultured in medium with addition of auxin and cytokinin, the 2HA explants form embryos. Generally cv Jemalong does not form embryos but does produce early vascularisation in the calli. The pasture legume M. truncatula (Australian barrel medic) is one of the model systems for the analysis of the unique biological and fundamental processes governing legume biology. Recent genomic tools, advanced DNA sequencing programs, EST libraries and Medicago GeneChip® have been developed for this legume and we previously have established proteome reference maps for M. truncatula somatic embryogenesis cultures and compared the proteome of the super-embryogenic line 2HA with that of non-embryogenic progenitor Jemalong [5, 36]. In this study, we have used leaf explant tissue cultures of 2HA and Jemalong to investigate gene expression profiles and their changes during the early stage of regeneration and to identify key regulatory factors and the early markers of cell competency for regeneration.
Transcriptomic analysis of the super-embryogenic line 2HA and its progenitor Jemalong
Comparison of real-time RT-PCR and microarray results for selected genes
1.43 ± 0.28
4.22 ± 0.53
RNA-directed DNA polymerase
1.37 ± 0.14
4.39 ± 0.89
1.32 ± 0.43
6.92 ± 0.58
1.10 ± 0.30
2.77 ± 0.17
1.06 ± 0.10
4.84 ± 0.16
1.32 ± 0.14
3.33 ± 0.37
1.19 ± 0.34
3.10 ± 0.53
-1.41 ± 0.11
-2.76 ± 0.09
1.71 ± 0.08
6.24 ± 0.28
1.52 ± 0.02
3.9 ± 0.03
Functional classification of differentially expressed probe sets
Potential metabolic differences in embryogenic and non-embryogenic cultures.
Embryogenic 2HA culture
No. E.C. numbers in genome array pathway
No. E.C. numbers expressed ≥ 2.0 fold
Stilbene, coumarine and lignin biosynthesis
Biosynthesis of 12-, 14- and 16-membered macrolides
Non-embryogenic Jemalong culture
Ascorbate and aldarate metabolism
Biosynthesis of 12-, 14- and 16-membered macrolides
We also annotated the array by comparing the data set with the Arabidopsis Gene Family Information database maintained by the Arabidopsis Information Resource . As of April 2007 the database contained 996 gene families and 8,331 genes. Using Blast, we were able to classify 3,159 Medicago probe sets into these families. Forty one and ten of the over expressed probe sets from the embryogenic and non-embryogenic cultures respectively were classified in the gene families. Two cytochrome P450 families (CYP94C, p = 0.016 and CYP90B, p = 0.047) were significantly over-represented in the non-embryogenic line Jemalong (Additional file 4). Finally, transcription factors (TFs) on the Genome array were predicted by homology relationship based on the Database of Arabidopsis Transcription Factors . This analysis showed that 2,323 probe sets on the Genome array have sequence homology to described plant TFs. Twenty one predicted TFs were up-regulated in the embryogenic line 2HA cultures and six TFs were up-regulated in the non-embryogenic Jemalong cultures (Table 4 and Additional file 5). The families represented in the embryogenic cultures are the basic/helix-loop-helix (bHLH), zinc finger domain TFs C2C2-co-like and C2C2-DOF, response regulators (GARP-ARR-B), GRAS domain containing TFs (GRAS), MADS-box TFs (MADS) and MYB DNA-binding domain TFs (MYB). The TF families represented in the non-embryogenic cultures are APETALA 2 and ethylene-responsive element binding proteins (AP2/EREBP), auxin-responsive protein/indoleacetic acid-induced protein (AUX/IAA) and ETHYLENE INSENSITIVE 3 (EIN3). With the exception of bHLH and zinc finger containing TFs, the TF gene families are plant specific. We confirmed the expression of several TFs betweens the cultures of two lines using qRT-PCR (Table 1).
Phytohormone biosynthesis and signalling
Although GeneBins and PathExpress are valuable tools to identify gene classes and molecular pathways in general, they are not designed to identify plant specific pathways such as phytohormone biosynthesis and signalling. Thus, we manually analysed the differentially displayed genes involved in these processes. We have identified two probe sets Mtr.30770.1.S1_at & Mtr.10439.1.S1_at that are homologues to Arabidopsis ETHYLENE INSENSITIVE3 (EIN3). These two probe sets were down-regulated 2.6 fold and 1.8 fold respectively, in the embryogenic 2HA cultures. Similarly, a probe set for GA2-oxidase (GA2ox) (Mtr.33914.1.S1_at) and a probe set (Mtr.22904.1.S1_s_at) for an IAA/AUX gene was down-regulated in the embryogenic 2HA cultures. In contrast, a response regulator (MtRR1, Mtr.43735.1.S1_at) was up-regulated the embryogenic 2HA cultures. This was confirmed by real-time RT-PCR (Table 1).
Comparison of gene expression between the embryogenic cultures and seed development
To identify common genes expressed between embryogenic cultures (somatic embryogenesis) and developing seeds (zygotic embryogenesis), we have compared our data to that from the Medicago Expression Atlas . We have chosen developing seeds at 10 days after pollination since this is the earliest time point available for seed development in the Atlas and contrasted it to leaf. A total of 12,954 probe sets showed differentially display between the developing seed at ten days after pollination and leaf samples. Over 6,800 probe sets were up-regulated in the developing seeds at least two fold (P < 0.05), of which 14 were also up-regulated in the embryogenic cultures when compared to non-embryogenic cultures (additional files 6 and 7). These include a basic helix-loop-helix (bHLH) transcription factor (Mtr.51379.1.S1_at), MtRR1 (response regulator, Mtr.43735.1.S1_at), a putative phosphatase (Mtr.10566.1.S1_at), an E1-E2 type ATPase (Mtr.26397.1.S1_s_at), a serine carboxypeptidase (Mtr.10023.1.S1_at), a GDSL-motif lipase (Mtr.13241.1.S1_at), a peroxidase (Mtr.10375.1.S1_at), two nodulins (Mtr.11717.1.S1_at and Mtr.41025.1.S1_at), a fatty acid elongase (Mtr.49305.1.S1_at) and four unknown proteins (Mtr.35655.1.S1_at, Mtr.18491.1.S1_at, Mtr.38330.1.S1_at and Mtr.14656.1.S1_at). Over 6,000 probe sets were down-regulated in the developing seeds at least two fold (P < 0.05), of which 6 were also down-regulated in the embryogenic cultures when compared to non-embryogenic cultures (additional files 6 and 7). these include transcription factor EIL1 (Mtr.10439.1.S1_at), a H+-transporting ATPase Mtr.5635.1.S1_at), Snakin-like cysteine rich protein (Mtr.12742.1.S1_at), a patatin-like phospholipase (Mtr.37859.1.S1_at), a thaumatin-like protein (Mtr.33691.1.S1_at) and a hypothetical protein (Mtr.43627.1.S1_at). In brief, we have identified a small number probe sets that were either up- or down-regulated in both the embryogenic cultures and the developing seeds. These include transcription factors such as response regulator MtRR1 and EIN3, nodulins and unknown proteins (additional file 6). Further investigation of these proteins will shed light on the similarities between somatic and zygotic embryogenesis.
Comparison between the array and proteomics
We also compared our array data with the proteome data obtained for the explant leaf cultures of 2HA and Jemalong . 16 protein spots were reportedly identified as differentially displayed proteins between the explant leaf cultures of 2HA and Jemalong after 2, 5 and 8 weeks of culture. Although all of the corresponding genes were present on the array, none of them showed differential display when used 2 fold cut-off and student t test (data not shown). Thus, we were not able to find any correlation between transcriptomics and proteomics of the explant leaf cultures of 2HA and Jemalong. This probably due to the fact that only a very limited number of differentially displayed proteins were identified by proteomics, most of which showed differential display only at the later stages of culture (5 and 8 weeks of culture) but not at the early stage (at two weeks) at which this microarray analysis was focused on.
During the initial phases of organogenesis somatic cells progress through a series of events referred to as differentiation, competence acquisition, induction and determination . Most in vitro cultures require auxin in the medium to initiate these steps while sunflower immature zygotic embryos do not. They do, however require cytokinin to induce somatic embryogenesis [20, 45]. Working with immature zygotic embryos of sunflowers, Thomas et al. showed that the time of exposure to a specific medium was fundamental to the commitment to a particular morphogenic pathway . This period was described as embryogenic competence during the morphogenic induction. The period lasted for three days when the commitment could be reversed by changing the medium. However, after four days it could not be altered and thus an irreversible step was taken within the competent cells toward a particular organogenesis pathway. Seven days of pre-treatment with auxin can interrupt somatic embryo formation in M. truncatula . And at two weeks, the explant leaves start to proliferate. Thus, we reasoned that comparing transcriptomes of two-week old tissue cultures of super-embryogenic 2HA and its non-embryogenic progenitor Jemalong would reveal important genes involved in early steps of regeneration and acquiring totipotency. The transcriptomic analysis has revealed changes in gene expression between the super-embryogenic line and the non-embryogenic line of M. truncatula, although the vast majority of probe sets (over 99.5%) did not show any significant change between the cultures. The differentially expressed genes include genes involved in various metabolic pathways, flavonoid biosynthesis, hormone biosynthesis and signalling and genes involved in gene regulation.
We have identified five probe sets (Mtr.18380.1.S1_at, Mtr.10992.1.S1_at, Mtr.17361.1.S1_at, Mtr.51607.1.S1_at and Mtr.50900.1.S1_at) belonging to Beta-Ig-H3 fasciclin-like arabinogalactan proteins (AGPs) that are up-regulated in the embryogenic cultures of 2HA at least two fold. AGPs are implicated in diverse developmental roles including somatic embryogenesis  although their exact functions remain unclear. AGPs containing N-acetylglucosamine can be a substrate for chitinase  leading to the release of oligosaccharide signal molecules that are necessary to induce somatic embryo formation . The involvement of extracellular signal molecules in somatic embryogenesis has been reported in several plant species. It was shown that when non-embryogenic cultures were treated with growth medium conditioned by super-embryogenic cultures, the cultures became embryogenic . Several components in the conditioned growth medium have been found to promote somatic embryogenesis. These components include chitinases  and AGPs [51–54]. It has been suggested that oligosaccharides released from AGPs by a chitinase act as signal molecules stimulating somatic embryogenesis . However, the role of AGPs in the induction of somatic embryogenesis in M. truncatula is not understood yet.
Genes involved in transition from vegetative growth to reproductive growth
We have identified an Arabidopsis ortholog of FLOWERING PROMOTING FACTOR1 (AtFPF1) that was 2.3 fold up-regulated in 2HA (Mtr.41073.1.S1_at). AtFPF1 is one of the important genes involved in the genetic control of flowering time in Arabidopsis. It is expressed in apical meristems immediately after photoperiodic induction of flowering in long-day plants, which flower only when exposed to long days . During the transition to flowering, the FPF1 gene is expressed at the same time as LEAFY and earlier than APETALA1, two key unrelated TFs in flower initiation. FPF1 modulates the acquisition of competence to flower in the apical meristem. Over-expression of FPF1 leads to early flowering in Arabidopsis . Similar results were also reported in tobacco . However in rice, it has been shown that it also plays a role in the initiation of adventitious roots [59, 60] and it has been reported that the same gene was induced by salt treatments in M. truncatula roots and may contribute to the reacquisition of root growth, notably through the emergence of lateral roots . Another flowering promoting gene that was up-regulated (2.3 fold) in 2HA is AGAMOUS-LIKE 20 (AGL20, also known as SUPPRESSOR OF OVEREXPRESSION OF CO 1 or SOC1, Mtr.47174.1.S1_at) encodes a MADS box TF. In Arabidopsis, its ortholog was identified as a gene downstream of another MADS box TF FLC . Activation of AGL20 causes early flowering despite strong expression of FLC, and knock out of AGL20 causes late flowering, suggesting that it is a flowering activator . AGL20 is positively regulated by the long day pathway through CO, and negatively regulated by the autonomous/vernalisation pathway through FLC [62, 63]. Since expression of AGL20 is regulated by signals from more than one flowering pathway it is referred to as a floral pathway integrator [64, 65]. These genes function in 'cascades' within four promotive pathways, the 'photoperiodic', 'autonomous', 'vernalisation', and 'gibberellin' pathways, which all converge on the 'integrator' genes AGL20 (SOC1) and FLOWERING LOCUS T (FT) . It has been shown that FLC directly interacts with the AGL20 and FT genes in vivo . Probe set Mtr.7513.1.S1_at was up-regulated in 2HA and encodes a CONSTANS-like TF that are ortholog of At1g25440, which displayed root-specific expression  and are strongly repressed in N starvation  suggesting biological functions beyond promoting flowering.
Thus, we have identified three genes that were up-regulated in 2HA have similarities to the genes involved in transition from vegetative growth to reproductive growth, suggesting that initiation of both reproductive growth and regeneration share similar molecular processes.
We identified eight genes classified as nodulins including early nodulin 75 (Mtr.38422.1.S1_at), MtN3s (Mtr.8585.1.S1_at & Mtr.11146.1.S1_at), MtN13 (Mtr.33137.1.S1_s_at & Mtr.37852.1.S1_at), nodulin 26 (Mtr.36842.1.S1_s_at) and other nodulins Mtr.43745.1.S1_at & Mtr.43508.1.S1_at). MtN3 protein contains MtN3 and saliva related transmembrane protein domain (Mtr.8585.1.S1_at & Mtr.11146.1.S1_at) and reported to be induced during nodulation in M. truncatula . It has been shown in ascidian Ciona intestinalis that a gene encoding an MtN3/saliva family transmembrane protein is essential for tissue differentiation during embryogenesis . MtN13, a homologue of plant defence proteins (Pathogenesis-related protein Bet v I family) has been reported to be nodulation/symbiosis-specific in M. truncatula . Nod26, a member of plant aquaporins, also has been shown to be involved in nodulation [72, 73]. Another non-nodulin proteins that has shown to be involved in nodule development is cycloartenol synthase . We have detected the same gene (Mtr.4710.1.S1_s_at) highly up-regulated in the embryogenic line 2HA. These indicate that several genes expressed during nodule formation also expressed during regeneration in M. truncatula.
Phytohormone biosynthesis and signalling
Two probe sets Mtr.10439.1.S1_at & Mtr.30770.1.S1_at that are homologues to Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) were down-regulated 2.6 fold and 1.8 fold respectively, in the embryogenic line 2HA. The probe set Mtr.10439.1.S1_at was also down-regulated in the developing seeds at 10 days after pollination when compared to leaf samples, indicating some similarities between somatic and zygotic embryogenesis. EIN3 acts as a positive regulator at the most downstream position of the ethylene signal transduction pathway . EIN3 encodes a transcription factor that belongs to a small family that includes EIN3 and various EIN3-like (EIL) proteins in Arabidopsis and it works downstream of EIN2  and upstream of AtERF1, an early ethylene responsive gene . Recently, Achard et al. has shown that activated ethylene signalling reduces bioactive Gibberellin (GA) levels and enhances the accumulation of DELLAs, and ethylene acts on DELLAs via the CTR1-dependent ethylene response pathway, most likely downstream of the transcriptional regulator EIN3. Ethylene-enhanced DELLA accumulation in turn delays flowering via repression of the floral meristem-identity genes LEAFY and AGL20 (SOC1), establishing a link between the CTR1/EIN3-dependent ethylene and GA-DELLA signalling pathways .
We have observed that a probe set for GA2-oxidase (GA2ox) (Mtr.33914.1.S1_at) was up-regulated in the non-embryogenic Jemalong cultures. GA has been implied to have an role in somatic embryogenesis in carrots , in Arabidopsis  and in Japanese cedar . GA2ox, introduces a hydroxyl group at the 2β position, inactivating the GA molecule so that it cannot be converted into active forms [80, 81]. These indicate that there is a reduction in active GA in this the non-embryogenic line Jemalong. However, the measuring of active GA contents in these lines is required to confirm such indication. It has been shown in Arabidopsis that AGL20 (or SOC1) is induced by GA  and we found AGL20 (SOC1, Mtr.47174.1.S1_at) to be up-regulated in the embryogenic line 2HA. The up-regulation of AGL20 correlates well with the up-regulation of GA2ox and down-regulation of EIN3 in the embryogenic line. Thus, our findings suggest that GA and ethylene may be involved in the acquisition of regeneration capacity in M. truncatula and indicate that AGL20 may be a key regulator that links GA and ethylene signalling.
We have identified a probe set (Mtr.22904.1.S1_s_at) for an IAA/AUX gene that was down-regulated in the embryogenic cultures. The corresponding gene is an ortholog of Arabidopsis IAA20 (AT2G46990). In Arabidopsis, IAA20 protein is long-lived and its longevity was not influenced by auxin suggesting they may play a novel role in auxin signalling . We previously have shown that auxin (1-naphthaleneacetic acid) pre-incubation explant leaf tissues can irreversibly interrupt somatic embryo formation in the M. truncatula embryogenic line 2HA . Thus, up-regulation of IAA20 ortholog in M. truncatula supports motion that the prolonged auxin signalling may have adverse effect on embryo formation.
Proliferation of undifferentiated callus tissue, greening, and the formation of shoot structures are all cytokinin-dependent processes. We have identified a response regulator (MtRR1, Mtr.43735.1.S1_at) that is up-regulated in the embryogenic cultures. This probe set was also up-regulated in the developing seeds at 10 days after pollination when compared to leaf samples, indicating some similarities between somatic and zygotic embryogenesis. MtRR1 is an ortholog of Arabidopsis ARR10 (RESPONSE REGULATOR 10; At4g31920) that belongs to B-type response regulators. It was reported that this gene is induced early in M. truncatula roots during the symbiotic interaction with Sinorhizobium meliloti . There are other probe sets for the genes involved in cytokinin biosynthesis and signalling. However, these were not changed between the two cultures. For instance, there are two probe sets for adenylate isopentenyltransferases (cytokinin synthases, Mtr.31420.1.S1_at & Mtr.12113.1.S1_at) in the array and both probe sets did not expressed in both cultures. In contrast, Cytokinin Response 1, (CRE1, Mtr.12088.1.S1_at)  and other cytokinin inducible genes cyclin D3 (Mtr.35281.1.S1_at and Mtr.41123.1.S1_at), KNAT (Mtr.8842.1.S1_at), SHOOT MERISTEMLESS (Mtr.13772.1.S1_at) and type A response regulators (cytokinin-inducible) (Mtr.5343.1.S1_s_at, Mtr.32159.1.S1_at, Mtr.5335.1.S1_at, Mtr.43919.1.S1_at, Mtr.31738.1.S1_at and Mtr.174.1.S1_at) were also highly expressed in both cultures. These indicate that there are some differences between the embryogenic line 2HA and the non-embryogenic line Jemalong in respond to cytokinin and MtRR1 may be an important regulator in the acquisition of regeneration capacity in M. truncatula.
We have described differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong. Notably they include significant variations in carbon and flavonoid metabolism, phytohormone biosynthesis and signalling, cell to cell communication and gene regulation. This data will facilitate the mapping of regulatory and metabolic networks involved in the acquisition of regeneration capacity of the embryogenic lines such as 2HA, and may lead to a better understanding of totipotency in M. truncatula and other legume species.
Plant materials, growth and tissue culture
M. truncatula cv Jemalong seed line 2HA and its progenitor Jemalong was used for the plant growth explant tissue culture as described [36, 85]. Seeds of M. truncatula cv Jemalong were obtained from Professor Ray Rose (University of Newcastle, NSW, Australia). Plants were grown under controlled growth cabinet conditions with 12 hr photoperiod at 150 μmol m-2 s-1 with a day temperature of 23°C and a night temperature of 19°C and a relative humidity of 80%. The basal medium used for the explant leaf culture was P4, which is based on Gamborg's B5 medium as described . In the usual culture procedure, leaf explants were plated onto P4 medium containing 10 μM NAA (1-naphthaleneacetic acid, Sigma-Aldrich, St. Louis, MO, USA) and 4 ╀M BAP (6-benzylaminopurine, Sigma-Aldrich). Cultures were incubated in the dark at 28°C.
DNA microarray analysis
The Affymetrix Medicago GeneChip (Affymetrix, Santa Clara, CA, USA) contained 61,200 probe sets: 32,167 M. truncatula EST-based and chloroplast gene-based probe sets (TIGR Gene Index version 8, Jan., 2005, 36,878 unique sequences); 18,733 M. truncatula IMGAG (International Medicago Genome Annotation Group) and phase 2/3 BAC prediction-based probe sets; 1,896 M. sativa EST/mRNA based probe sets; 8,305 Sinorhizobium meliloti gene prediction-based probe sets.
RNA isolation, hybridisation and data pre-processing
Total RNA was extracted and purified from the proliferating leaf explant cultures of M. truncatula line 2HA and Jemalong using the Qiagen RNeasy plant mini kit (Qiagen, Valencia, CA, USA). Total RNA was quantified using a NanoDrop ND-1000 Spectrophotometer; RNA with an absorbance A260/A280ratio >2.0 was quality tested using the Agilent 2100 Bioanalyzer. Preparation of cRNA, hybridisation, and scanning of the Test3 arrays and Medicago GeneChip® were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA, USA) (at the Biomolecular Resource Facility, JCSMR, ANU). Briefly, double-stranded cDNA was synthesised from 5 to 8 μg of each RNA sample via oligo T7-(dT)24 primer-mediated reverse transcription. Biotin-labelled cRNA was generated using the Enzo BioArray kit (Affymetrix), purified using RNeasy spin columns (Qiagen), and then quantified by spectrophotometer. Fifteen to 20 μg of each biotin-labelled fragmented cRNA sample was used to prepare 300 μL of hybridisation mixture. Aliquots of each sample (100 μL) were hybridised onto Test3 arrays to check the quality of the samples prior to hybridisation (200 μL) onto the Medicago genome arrays. The arrays were washed with optimised wash protocols, stained with strepdavidin/phycoerythrin followed by antibody amplification, and scanned with the Agilent GeneArray Scanner (Affymetrix).
Raw Affymetrix data (cel files) were normalised with the GCRMA (GC content – Robust Multi-Array Average) algorithm (ver. 2.2.0) including quantile normalisation and variance stabilisation , using the Affymetrix package of the bioconductor software . The normalised average of the replicates was then log transformed in base 2 to reduce the proportional relationship between random error and signal intensity. Differentially expressed probe sets were identified by evaluating the log2 ratio between the two conditions associated to a standard t-test , adjusted for multiple testing by the False Discovery Rate (FDR) approach . All probe sets that differed more than to two-fold with a t-test P-value ≤ 0.05 were considered to be differentially expressed. The Significance Analysis of Microarrays (SAM) two-class unpaired analysis  was also performed in order to identify a more extensive list of differentially expressed genes, with the measure significant fold change set at 2.0 and a false discovery rate <8.4%. The expected proportion of significantly different features (p0) was set to 0.95.
Functional categories significantly associated (P-value ≤ 0.05, adjusted using the FDR correction) with the up- and down-regulated sequences were identified using GeneBins, a database that provides a hierarchical functional classification modelled on the KEGG ontology  of probe set sequences represented on Affymetrix arrays . We used PathExpress , a web-based tool based on the KEGG Ligand database , to detect whether probe sets associated with a metabolic pathway or sub-pathway were statistically over-represented in the differentially expressed sets of sequences (P-value ≤ 0.05). In addition, probe sets of the Affymetrix Medicago Genome Array were assigned to gene families described in the TAIR database  and to transcription factor families provided by the Database of Arabidopsis Transcription Factors  based on their sequence similarity with Arabidopsis thaliana proteins. Blastx  was used to find the best match (E-value ≤ 10-8) for the sequences representing each probe set (i.e. sequences derived from the most 5' to the most 3' probe in the public UniGene cluster). The differentially expressed sets of sequences were compared to the composition of each gene family to identify if a certain category was statistically over-represented. For each test, a P-value, representing the probability that the intersection of the list of up- or down-regulated probe sets with the list of probe sets belonging to the given gene family occurs by chance, was calculated using the hypergeometric distribution .
Sequences of interest were analysed using BLAST and multiple sequence alignments to identify genes and proteins with sequence similarity from Arabidopsis. To identify orthologs in Arabidopsis, AffyTrees was used http://bioinfoserver.rsbs.anu.edu.au/utils/affytrees/. AffyTrees automatically detects sequence orthologs based on phylogenetic trees.
Comparing to Medicago Expression Atlas
To identify common genes expressed between embryogenic cultures and developing seeds, we have compared our data to that of the Medicago Expression Atlas . We have chosen seed10d (Developing seeds at early embryogenesis – 10 days after pollination) since it is the earliest time point for seed development available in the Atlas and contrasted this to leaf (4-week old trifolia that were harvested without their petioles but with their petiolule) and have computed the average between all replicates, ratios (seed/leaf), log2 (ratio), t test adjusted with FDR method). Then we compared these lists with our data to see any overlap.
Primers used in real-time RT-PCR assay
RNA-directed DNA polymerase
Not on the array
Transcription factor families that are different between the embryogenic and the non-embryogenic cultures.
Number of probe sets on array
≥ 2 fold up in embryogenic culture
≥ 2 fold up in non-embryogenic culture
1 (p = 0.126)
1 (p = 0.024)
5 (p = 0.005)
1 (p = 0.140)
1 (p = 0.654)
2 (p = 0.179)
2 (p = 0.016)
1 (p = 0.480)
1 (p = 0.006)
1 (p = 0.071)
1 (p = 0.251)
1 (p = 0.188)
2 (p = 0.193)
6 (p = 0.106)
1 (p = 0.556)
We thank Ray Rose for providing Jemalong and 2HA seeds, Jeff Wilson for the photograph and Kaiman Peng at the John Curtin School of Medical Research, ANU for processing the Affymetrix GeneChips. We also would like to thank Peta Holmes for her contribution. This work was supported by the Australian Research Council (Grant No CEO348212).
- Takebe I, Labib G, Melchers G: Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwissenschaften. 1971, 58: 318-320. 10.1007/BF00624737.View ArticleGoogle Scholar
- Zimmerman LJ: Somatic embryogenesis: A model for early development in higher plants. Plant Cell. 1993, 5: 1411-1423. 10.1105/tpc.5.10.1411.PubMedPubMed CentralView ArticleGoogle Scholar
- Schmidt EDL, Guzzo F, Toonen MAJ, Devries SC: A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development. 1997, 124 (10): 2049-2062.PubMedGoogle Scholar
- Nolan KE, Irwanto RR, Rose RJ: Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures. Plant Physiol. 2003, 133 (1): 218-230. 10.1104/pp.103.020917.PubMedPubMed CentralView ArticleGoogle Scholar
- Imin N, De Jong F, Mathesius U, van Noorden G, Saeed NA, Wang XD, Rose RJ, Rolfe BG: Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts. Proteomics. 2004, 4 (7): 1883-1896. 10.1002/pmic.200300803.PubMedView ArticleGoogle Scholar
- Giroux RW, Pauls KP: Characterization of somatic embryogenesis-related cDNAs from alfalfa (Medicago sativa L.). Plant Mol Biol. 1997, 33 (3): 393-404. 10.1023/A:1005786826672.PubMedView ArticleGoogle Scholar
- Stasolla C, Bozhkov PV, Chu TM, Van Zyl L, Egertsdotter U, Suarez MF, Craig D, Wolfinger RD, Von Arnold S, Sederoff RR: Variation in transcript abundance during somatic embryogenesis in gymnosperms. Tree Physiol. 2004, 24 (10): 1073-1085.PubMedView ArticleGoogle Scholar
- van Zyl L, Bozhkov PV, Clapham DH, Sederoff RR, von Arnold S: Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis. Gene Expr Patterns. 2003, 3 (1): 83-91. 10.1016/S1567-133X(02)00068-6.PubMedView ArticleGoogle Scholar
- Stasolla C, van Zyl L, Egertsdotter U, Craig D, Liu W, Sederoff RR: The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos. Plant Physiol. 2003, 131 (1): 49-60. 10.1104/pp.015214.PubMedPubMed CentralView ArticleGoogle Scholar
- van Zyl L, von Arnold S, Bozhkov P, Chen Y, Egertsdotter U, MacKay J, Sederoff R, Shen J, Zelena L, Clapham D: Heterologous array analysis in Pinaceae: Hybridization of high density arrays of Pinus taeda cDNA from needles and embryogenic cultures of P. taeda, P. sylvestris or Picea abies. Comp Funct Genom. 2002, 3: 306-318. 10.1002/cfg.199.View ArticleGoogle Scholar
- Malik MR, Wang F, Dirpaul JM, Zhou N, Polowick PL, Ferrie AM, Krochko JE: Transcript profiling and identification of molecular markers for early microspore embryogenesis in Brassica napus. Plant Physiol. 2007, 144 (1): 134-154. 10.1104/pp.106.092932.PubMedPubMed CentralView ArticleGoogle Scholar
- Le BH, Wagmaister JA, Kawashima T, Bui AQ, Harada JJ, Goldberg RB: Using genomics to study legume seed development. Plant Physiol. 2007, 144 (2): 562-574. 10.1104/pp.107.100362.PubMedPubMed CentralView ArticleGoogle Scholar
- Chugh A, Khurana P: Gene expression during somatic embryogenesis – recent advances. Current Science. 2002, 86: 715-730.Google Scholar
- Mordhorst AP, Toonen MAJ, Devries SC: Plant embryogenesis. Crit Rev Plant Sci. 1997, 16 (6): 535-576. 10.1080/713608156.View ArticleGoogle Scholar
- Walker JC, Key JL: Isolation of Cloned cDNAs to Auxin-Responsive Poly(A)+RNAs of Elongating Soybean Hypocotyl. Proc Natl Acad Sci USA. 1982, 79: 7185-7189. 10.1073/pnas.79.23.7185.PubMedPubMed CentralView ArticleGoogle Scholar
- Yang H, Saitou T, Komeda Y, Harada H, Kamada H: Arabidopsis thaliana ECP63 encoding a LEA protein is located in chromosome 4. Gene. 1997, 184 (1): 83-88.PubMedGoogle Scholar
- Overvoorde PJ, Grimes HD: The role of calcium and calmodulin in carrot somatic embryogenesis. Plant Cell Physiol. 1994, 35: 135-144.Google Scholar
- Lindzen E, Choi JH: A carrot cDNA encoding an atypical protein kinase homologous to plant calcium-dependent protein kinases. Plant Mol Biol. 1995, 28 (5): 785-797. 10.1007/BF00042065.PubMedView ArticleGoogle Scholar
- Davletova S, Meszaros T, Miskolczi P, Oberschall A, Torok K, Magyar Z, Dudits D, Deak M: Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells. J Exp Bot. 2001, 52 (355): 215-221. 10.1093/jexbot/52.355.215.PubMedView ArticleGoogle Scholar
- Thomas C, Meyer D, Himber C, Steinmetz A: Spatial expression of a sunflower SERK gene during induction of somatic embryogenesis and shoot organogenesis. Plant Physiol Biochem. 2004, 42 (1): 35-42. 10.1016/j.plaphy.2003.10.008.PubMedView ArticleGoogle Scholar
- Kawahara R, Komamine A, Fukuda H: Isolation and characterization of homeobox-containing genes of carrot. Plant Mol Biol. 1995, 27 (1): 155-164. 10.1007/BF00019187.PubMedView ArticleGoogle Scholar
- Meijer AH, Scarpella E, van Dijk EL, Qin L, Taal AJ, Rueb S, Harrington SE, McCouch SR, Schilperoort RA, Hoge JH: Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice. Plant J. 1997, 11 (2): 263-276. 10.1046/j.1365-313X.1997.11020263.x.PubMedView ArticleGoogle Scholar
- De Jong AJ, Cordewener J, Lo Schiavo F, Terzi M, Vandekerckhove J, Van Kammen A, De Vries SC: A carrot somatic embryo mutant is rescued by chitinase. Plant Cell. 1992, 4 (4): 425-433. 10.1105/tpc.4.4.425.PubMedPubMed CentralView ArticleGoogle Scholar
- Baldwin TC, Domingo C, Schindler T, Seetharaman G, Stacey N, Roberts K: DcAGP1, a secreted arabinogalactan protein, is related to a family of basic proline-rich proteins. Plant Mol Biol. 2001, 45 (4): 421-435. 10.1023/A:1010637426934.PubMedView ArticleGoogle Scholar
- Sterk P, Booij H, Schellekens GA, Van Kammen A, De Vries SC: Cell-specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell. 1991, 3 (9): 907-921. 10.1105/tpc.3.9.907.PubMedPubMed CentralView ArticleGoogle Scholar
- Zuo JR, Niu QW, Frugis G, Chua NH: The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis. Plant J. 2002, 30 (3): 349-359. 10.1046/j.1365-313X.2002.01289.x.PubMedView ArticleGoogle Scholar
- Meinke DW: A homoeotic mutant of Arabidopsis thaliana with leafy cotyledons. Science. 1992, 258: 1647-1650. 10.1126/science.258.5088.1647.PubMedView ArticleGoogle Scholar
- Meinke DW, Franzmann LH, Nickle TC, Yeung EC: Leafy cotyledon mutants of Arabidopsis. Plant Cell. 1994, 6 (8): 1049-1064. 10.1105/tpc.6.8.1049.PubMedPubMed CentralView ArticleGoogle Scholar
- Avivi Y, Morad V, Ben-Meir H, Zhao J, Kashkush K, Tzfira T, Citovsky V, Grafi G: Reorganization of specific chromosomal domains and activation of silent genes in plant cells acquiring pluripotentiality. Dev Dyn. 2004, 230 (1): 12-22. 10.1002/dvdy.20006.PubMedView ArticleGoogle Scholar
- Somleva MN, Schmidt EDL, de Vries SC: Embryonic cells in Dactylis glomerata L. (Poaceae) explants identified by cell tracking and by SERK expression. Plant Cell Reports. 2000, 19: 718-726. 10.1007/s002999900169.View ArticleGoogle Scholar
- Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt ED, Boutilier K, Grossniklaus U, de Vries SC: The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol. 2001, 127 (3): 803-816. 10.1104/pp.127.3.803.PubMedPubMed CentralView ArticleGoogle Scholar
- Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang LM, Hattori J, Liu CM, van Lammeren AAM, Miki BLA, Custers JBM, Campagne MMV: Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell. 2002, 14 (8): 1737-1749. 10.1105/tpc.001941.PubMedPubMed CentralView ArticleGoogle Scholar
- Somers DA, Samac DA, Olhoft PM: Recent advances in legume transformation. Plant Physiol. 2003, 131 (3): 892-899. 10.1104/pp.102.017681.PubMedPubMed CentralView ArticleGoogle Scholar
- Rose RJ, Nolan KE: Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants. Plant Cell Reports. 1995, 14 (6): 349-353. 10.1007/BF00238595.PubMedView ArticleGoogle Scholar
- Nolan KE, Rose RJ, Gorst JR: Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis from regenerated plants. Plant Cell Reports. 1989, 8: 278-281. 10.1007/BF00274129.PubMedView ArticleGoogle Scholar
- Imin N, Nizamidin M, Daniher D, Nolan KE, Rose RJ, Rolfe BG: Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments. Plant Physiol. 2005, 137 (4): 1250-1260. 10.1104/pp.104.055277.PubMedPubMed CentralView ArticleGoogle Scholar
- Holmes P, Goffard N, Weiller GF, Rolfe BG, Imin N: Transcriptional profiling of Medicago truncatula meristematic root cells. BMC Plant Biol. 2008, 8: 21-10.1186/1471-2229-8-21.PubMedPubMed CentralView ArticleGoogle Scholar
- Tesfaye M, Silverstein KAT, Bucciarelli B, Samac DA, Vance CP: The Affymetrix Medicago GeneChip® array is applicable for transcript analysis of alfalfa (Medicago sativa). Func Plant Biol. 2006, 33: 783-788. 10.1071/FP06065.View ArticleGoogle Scholar
- Research School of Biological Sciences. [http://bioinfoserver.rsbs.anu.edu.au/utils/GeneBins/]
- Goffard N, Weiller G: GeneBins: a database for classifying gene expression data, with application to plant genome arrays. BMC Bioinformatics. 2007, 8: 87-10.1186/1471-2105-8-87.PubMedPubMed CentralView ArticleGoogle Scholar
- Research School of Biological Sciences. [http://bioinfoserver.rsbs.anu.edu.au/utils/PathExpress/]
- Research School of Biological Sciences. [http://www.arabidopsis.org/browse/genefamily/index.jsp]
- Guo A, He K, Liu D, Bai S, Gu X, Wei L, Luo J: DATF: a database of Arabidopsis transcription factors. Bioinformatics. 2005, 21 (10): 2568-2569. 10.1093/bioinformatics/bti334.PubMedView ArticleGoogle Scholar
- Benedito VA, Torres-Jerez I, Murray JD, Andriankaja A, Allen S, Kakar K, Wandrey M, Verdier J, Zuber H, Ott T, Moreau S, Niebel A, Frickey T, Weiller G, He J, Dai X, Zhao PX, Tang Y, Udvardi MK: A gene expression atlas of the model legume Medicago truncatula. Plant J. 2008Google Scholar
- Dudits D, Bögre L, Györgyey J: Molecular and cellular approaches to the analysis of plant embryo development from somatic cells in-vitro. J Cell Sci. 1991, 99: 475-484.Google Scholar
- Imin N, Nizamidin M, Wu T, Rolfe BG: Factors involved in root formation in Medicago truncatula. J Exp Bot. 2007, 58 (3): 439-451. 10.1093/jxb/erl224.PubMedView ArticleGoogle Scholar
- Showalter AM: Arabinogalactan-proteins: structure, expression and function. Cell Mol Life Sci. 2001, 58 (10): 1399-1417. 10.1007/PL00000784.PubMedView ArticleGoogle Scholar
- Rojas-Herrera R, Loyola-Vargas VM: Induction of a class III acidic chitinase in foliar explants of Coffea arabica L. during somatic embryogenesis and wounding. Plant Sci. 2002, 163 (4): 705-711. 10.1016/S0168-9452(02)00156-5.View ArticleGoogle Scholar
- McCabe PF, Valentine TA, Forsberg LS, Pennell RI: Soluble signals from cells identified at the cell wall establish a developmental pathway in carrot. Plant Cell. 1997, 9 (12): 2225-2241. 10.1105/tpc.9.12.2225.PubMedPubMed CentralView ArticleGoogle Scholar
- Hari V: Effect of cell-density changes and conditioned media on carrot cell embryogenesis. Zeitschrift Fur Pflanzenphysiologie. 1980, 96 (3): 227-231.View ArticleGoogle Scholar
- Chapman A, Blervacq AS, Vasseur J, Hilbert JL: Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of beta-glucosyl Yariv reagent and epitope localisation during embryo development. Planta. 2000, 211 (3): 305-314. 10.1007/s004250000299.PubMedView ArticleGoogle Scholar
- Egertsdotter U, Vonarnold S: Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea-Abies). Physiologia Plantarum. 1995, 93 (2): 334-345. 10.1111/j.1399-3054.1995.tb02237.x.View ArticleGoogle Scholar
- Kreuger M, Vanholst GJ: Arabinogalactan-proteins are essential in somatic embryogenesis of Daucus-Carota L. Journal of Cellular Biochemistry. 1993, 19-19.Google Scholar
- Thompson HJM, Knox JP: Stage-specific responses of embryogenic carrot cell suspension cultures to arabinogalactan protein-binding beta-glucosyl Yariv reagent. Planta. 1998, 205 (1): 32-38. 10.1007/s004250050293.View ArticleGoogle Scholar
- van Hengel AJ, Tadesse Z, Immerzeel P, Schols H, van Kammen A, de Vries SC: N-acetylglucosamine and glucosamine-containing arabinogalactan proteins control somatic embryogenesis. Plant Physiol. 2001, 125 (4): 1880-1890. 10.1104/pp.125.4.1880.PubMedPubMed CentralView ArticleGoogle Scholar
- Kania T, Russenberger D, Peng S, Apel K, Melzer S: FPF1 promotes flowering in Arabidopsis. Plant Cell. 1997, 9 (8): 1327-1338. 10.1105/tpc.9.8.1327.PubMedPubMed CentralView ArticleGoogle Scholar
- Melzer S, Kampmann G, Chandler J, Apel K: FPF1 modulates the competence to flowering in Arabidopsis. Plant J. 1999, 18 (4): 395-405. 10.1046/j.1365-313X.1999.00461.x.PubMedView ArticleGoogle Scholar
- Smykal P, Gleissner R, Corbesier L, Apel K, Melzer S: Modulation of flowering responses in different Nicotiana varieties. Plant Mol Biol. 2004, 55 (2): 253-262. 10.1007/s11103-004-0557-8.PubMedView ArticleGoogle Scholar
- Ge L, Chen H, Jiang JF, Zhao Y, Xu ML, Xu YY, Tan KH, Xu ZH, Chong K: Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity. Plant Physiol. 2004, 135 (3): 1502-1513. 10.1104/pp.104.041996.PubMedPubMed CentralView ArticleGoogle Scholar
- Xu ML, Jiang JF, Ge L, Xu YY, Chen H, Zhao Y, Bi YR, Wen JQ, Chong K: FPF1 transgene leads to altered flowering time and root development in rice. Plant Cell Rep. 2005, 24 (2): 79-85. 10.1007/s00299-004-0906-8.PubMedView ArticleGoogle Scholar
- Merchan F, de Lorenzo L, Rizzo SG, Niebel A, Manyani H, Frugier F, Sousa C, Crespi M: Identification of regulatory pathways involved in the reacquisition of root growth after salt stress in Medicago truncatula. Plant J. 2007, 51 (1): 1-17. 10.1111/j.1365-313X.2007.03117.x.PubMedView ArticleGoogle Scholar
- Lee H, Suh SS, Park E, Cho E, Ahn JH, Kim SG, Lee JS, Kwon YM, Lee I: The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev. 2000, 14 (18): 2366-2376. 10.1101/gad.813600.PubMedPubMed CentralView ArticleGoogle Scholar
- Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G: Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science. 2000, 288 (5471): 1613-1616. 10.1126/science.288.5471.1613.PubMedView ArticleGoogle Scholar
- Mouradov A, Cremer F, Coupland G: Control of flowering time: interacting pathways as a basis for diversity. Plant Cell. 2002, 14 (Suppl): S111-130.PubMedPubMed CentralGoogle Scholar
- Simpson GG, Dean C: Arabidopsis, the Rosetta stone of flowering time?. Science. 2002, 296 (5566): 285-289. 10.1126/science.296.5566.285.PubMedView ArticleGoogle Scholar
- Corbesier L, Coupland G: The quest for florigen: a review of recent progress. J Exp Bot. 2006, 57 (13): 3395-3403. 10.1093/jxb/erl095.PubMedView ArticleGoogle Scholar
- Helliwell CA, Wood CC, Robertson M, James Peacock W, Dennis ES: The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J. 2006, 46 (2): 183-192. 10.1111/j.1365-313X.2006.02686.x.PubMedView ArticleGoogle Scholar
- Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK: Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J. 2004, 38 (2): 366-379. 10.1111/j.1365-313X.2004.02051.x.PubMedView ArticleGoogle Scholar
- Scheible WR, Morcuende R, Czechowski T, Fritz C, Osuna D, Palacios-Rojas N, Schindelasch D, Thimm O, Udvardi MK, Stitt M: Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol. 2004, 136 (1): 2483-2499. 10.1104/pp.104.047019.PubMedPubMed CentralView ArticleGoogle Scholar
- Gamas P, Niebel Fde C, Lescure N, Cullimore J: Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development. Mol Plant Microbe Interact. 1996, 9 (4): 233-242.PubMedView ArticleGoogle Scholar
- Hamada M, Wada S, Kobayashi K, Satoh N: Ci-Rga, a gene encoding an MtN3/saliva family transmembrane protein, is essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis. Differentiation. 2005, 73 (7): 364-376. 10.1111/j.1432-0436.2005.00037.x.PubMedView ArticleGoogle Scholar
- Dean RM, Rivers RL, Zeidel ML, Roberts DM: Purification and functional reconstitution of soybean nodulin 26. An aquaporin with water and glycerol transport properties. Biochem. 1999, 38 (1): 347-353. 10.1021/bi982110c.View ArticleGoogle Scholar
- Rivers RL, Dean RM, Chandy G, Hall JE, Roberts DM, Zeidel ML: Functional analysis of nodulin 26, an aquaporin in soybean root nodule symbiosomes. J Biol Chem. 1997, 272 (26): 16256-16261. 10.1074/jbc.272.26.16256.PubMedView ArticleGoogle Scholar
- Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR: Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell. 1997, 89 (7): 1133-1144. 10.1016/S0092-8674(00)80300-1.PubMedView ArticleGoogle Scholar
- Solano R, Stepanova A, Chao Q, Ecker JR: Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev. 1998, 12 (23): 3703-3714. 10.1101/gad.12.23.3703.PubMedPubMed CentralView ArticleGoogle Scholar
- Achard P, Baghour M, Chapple A, Hedden P, Straeten Van Der D, Genschik P, Moritz T, Harberd NP: The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristem-identity genes. Proc Natl Acad Sci USA. 2007, 104 (15): 6484-6489. 10.1073/pnas.0610717104.PubMedPubMed CentralView ArticleGoogle Scholar
- Mitsuhashi W, Toyomasu T, Masui H, Katho T, Nakaminami K, Kashiwagi Y, Akutsu M, Kenmoku H, Sassa T, Yamaguchi S, Kamiya Y, Kamada H: Gibberellin is essentially required for carrot (Daucus carota L.) somatic embryogenesis: dynamic regulation of gibberellin 3-oxidase gene expressions. Biosci Biotechnol Biochem. 2003, 67 (11): 2438-2447. 10.1271/bbb.67.2438.PubMedView ArticleGoogle Scholar
- Curaba J, Moritz T, Blervaque R, Parcy F, Raz V, Herzog M, Vachon G: AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis. Plant Physiol. 2004, 136 (3): 3660-3669. 10.1104/pp.104.047266.PubMedPubMed CentralView ArticleGoogle Scholar
- Igasaki T, Sato T, Akashi N, Mohri T, Maruyama E, Kinoshita I, Walter C, Shinohara K: Somatic embryogenesis and plant regeneration from immature zygotic embryos of Cryptomeria japonica D. Don. Plant Cell Rep. 2003, 22 (4): 239-243. 10.1007/s00299-003-0687-5.PubMedView ArticleGoogle Scholar
- Stavang JA, Lindgard B, Erntsen A, Lid SE, Moe R, Olsen JE: Thermoperiodic stem elongation involves transcriptional regulation of gibberellin deactivation in pea. Plant Physiol. 2005, 138 (4): 2344-2353. 10.1104/pp.105.063149.PubMedPubMed CentralView ArticleGoogle Scholar
- Thomas SG, Phillips AL, Hedden P: Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc Natl Acad Sci USA. 1999, 96 (8): 4698-4703. 10.1073/pnas.96.8.4698.PubMedPubMed CentralView ArticleGoogle Scholar
- Moon J, Suh SS, Lee H, Choi KR, Hong CB, Paek NC, Kim SG, Lee I: The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. Plant J. 2003, 35 (5): 613-623. 10.1046/j.1365-313X.2003.01833.x.PubMedView ArticleGoogle Scholar
- Dreher KA, Brown J, Saw RE, Callis J: The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness. Plant Cell. 2006, 18 (3): 699-714. 10.1105/tpc.105.039172.PubMedPubMed CentralView ArticleGoogle Scholar
- Gonzalez-Rizzo S, Crespi M, Frugier F: The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti. Plant Cell. 2006, 18 (10): 2680-2693. 10.1105/tpc.106.043778.PubMedPubMed CentralView ArticleGoogle Scholar
- Nolan KE, Rose RJ: Plant regeneration from cultured Medicago truncatula with particular references to abscisic acid and light treatments. Aust J Bot. 1998, 46 (1): 151-160. 10.1071/BT96138.View ArticleGoogle Scholar
- Thomas MR, Johnson LB, White FF: Selection of interspecific somatic hybrids of Medicago truncatula by using Agrobacterium-transformed tissues. Plant Sci. 1990, 69: 189-198. 10.1016/0168-9452(90)90117-7.View ArticleGoogle Scholar
- Wu Z, Irizarry RA, Gentleman R, Murillo FM, Spencer F: A model based background adjustment for oligonucleotide expression arrays. Technical Report. 2004, John Hopkins University, Department of Biostatistics Working Papers, Baltimore, MDGoogle Scholar
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5 (10): R80-10.1186/gb-2004-5-10-r80.PubMedPubMed CentralView ArticleGoogle Scholar
- Callow MJ, Dudoit S, Gong EL, Speed TP, Rubin EM: Microarray expression profiling identifies genes with altered expression in HDL-deficient mice. Genome Res. 2000, 10 (12): 2022-2029. 10.1101/gr.10.12.2022.PubMedPubMed CentralView ArticleGoogle Scholar
- Benjamini Y, Hochberg Y: Controlling the false discovery rate – a practical and powerful approach to multiple testing. J ROY STAT SOC B MET. 1995, 57 (1): 289-300.Google Scholar
- Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001, 98 (9): 5116-5121. 10.1073/pnas.091062498.PubMedPubMed CentralView ArticleGoogle Scholar
- Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M: The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004, D277-280. 10.1093/nar/gkh063. 32 DatabaseGoogle Scholar
- Goffard N, Weiller G: PathExpress: a web-based tool to identify relevant pathways in gene expression data. Nucleic Acids Research. 2007, July (35 Web Server): W176-W181. 10.1093/nar/gkm261.View ArticleGoogle Scholar
- Goto S, Okuno Y, Hattori M, Nishioka T, Kanehisa M: LIGAND: database of chemical compounds and reactions in biological pathways. Nucleic Acids Res. 2002, 30 (1): 402-404. 10.1093/nar/30.1.402.PubMedPubMed CentralView ArticleGoogle Scholar
- Rhee SY, Beavis W, Berardini TZ, Chen G, Dixon D, Doyle A, Garcia-Hernandez M, Huala E, Lander G, Montoya M, Miller N, Mueller LA, Mundodi S, Reiser L, Tacklind J, Weems DC, Wu Y, Xu I, Yoo D, Yoon J, Zhang P: The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 2003, 31 (1): 224-228. 10.1093/nar/gkg076.PubMedView ArticleGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410.PubMedView ArticleGoogle Scholar
- Cho RJ, Huang M, Campbell MJ, Dong H, Steinmetz L, Sapinoso L, Hampton G, Elledge SJ, Davis RW, Lockhart DJ: Transcriptional regulation and function during the human cell cycle. Nat Genet. 2001, 27 (1): 48-54. 10.1038/83751.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.