JXB Advance Access originally published online on December 6, 2006
Journal of Experimental Botany 2007 58(3):439-451; doi:10.1093/jxb/erl224
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Factors involved in root formation in Medicago truncatula
Australian Research Council Centre of Excellence for Integrative Legume Research, Genomic Interactions Group, Research School of Biological Sciences, Australian National University, Canberra City, ACT 2601, Australia
* To whom correspondence should be addressed. E-mail: nijat.imin{at}anu.edu.au
Received 3 July 2006; Revised 17 September 2006 Accepted 25 September 2006
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Abstract |
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The fact that auxin induces root formation has been known for more than half a century. However, despite the recent progress in this field, neither the molecular processes in which the auxin-responsive genes leading to root formation nor the interactions between phytohormones and other bioactive molecules during the commitment phase of root formation are well understood. Here the effect of biomolecules such as cytokinin, glutathione, and flavonoids, as well as the expression of several transcription factors in in vitro root formation in model legume Medicago truncatula are presented. It was demonstrated that auxin NAA (1-naphthaleneacetic acid) pretreatment for 7 d can irreversibly interrupt somatic embryo formation, whilst both reduced and oxidized forms of glutathione enhance root formation via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive skl mutant. It was also shown that quercetin and the well-known auxin transport inhibitor NPA (N-1-naphthylphthalamic acid), which has a similar structure to quercetin, and isoflavonoids formononetin and genistein caused severe reduction in root formation. Also, the relative expression of several transcription factors was analysed in 1-week-old NAA-treated explants (stem cell niche formation stage), in NAA- and BAP-treated explants (no root formation), and in the roots of germinated seeds. The results showed, for the first time in a legume, that the transcription factors homeodomain WOX5 and the AP2-domain containing PLETHORA1 and 2, BABY BOOM1 were strongly induced by auxin addition, while cytokinin addition dramatically reduced their expression, indicating a role for these genes in the formation of root stem cell niches.
Key words: Flavonoids, glutathione, Medicago truncatula, real-time RT-PCR, root formation, stem cell niche
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Introduction |
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One of the earliest findings of auxin activity was based on tissue culture studies, whereby auxin addition to undifferentiated callus tissue stimulated root formation (Skoog and Miller, 1957; Zimmerman, 1993). Subsequent investigations have shown that the plant hormone auxin is central to the control of plant growth and development. At the cellular level, auxin can regulate cell division and cell expansion and trigger specific differentiation events (Kepinski and Leyser, 2005; Woodward and Bartel, 2005). Auxins profoundly influence root morphology, inhibiting root elongation, increasing lateral root production, and along with the phytohormone cytokinin which induces shoot formation, auxin is basic to regeneration of plants from cultured callus (Krikorian, 1995). The control of root growth by auxin and cytokinin is a well-known example of hormone interactions in controlling plant development. Auxin plays a key role in promoting root growth, whereas cytokinin has an inhibitory effect. The outcome appears to depend on the ratio of the two hormones (for a review, Rashotte et al., 2005). The two plant hormones not only play opposite roles in controlling plant growth and development, but also influence each others’ hormone homeostasis (Binns et al., 1987; Palni et al., 1988; Bangerth, 1994; Zhang et al., 1995; Nordstrom et al., 2004). Cytokinin and auxin have antagonistic roles in root development: auxin promotes the formation of lateral roots (Malamy and Benfey, 1997; Zhang and Hasenstein, 1999; Casimiro et al., 2001; Guo et al., 2005) and adventitious roots (Falasca et al., 2004; Sorin et al., 2005), whereas cytokinin inhibits root formation at physiological concentrations and interferes with the auxin effect (Torrey, 1986; Zhang and Hasenstein, 1999; Lloret and Casero, 2002).
An important molecule in plant development is glutathione (reduced form, GSH; oxidized form, GSSG, also called glutathione disulphide), which is a ubiquitous tripeptide that is synthesized via two reactions catalysed by 
-glutamylcysteine synthesis and glutathione synthesis. Redox (oxidation–reduction) status is an important regulator of various metabolic functions in all eukaryotic cells. In plants, glutathione is a component of the ascorbate–glutathione cycle whereby it maintains the cellular redox homeostasis in response to oxidative stresses (Schafer and Buettner, 2001; Meyer and Hell, 2005). In this cycle, GSH is used by dehydroascorbate reductase to regenerate ascorbate which has the ability to scavenge H2O2. Here GSH is converted to GSSG, which is then regenerated by glutathione reductase (Asada and Takahashi, 1987; Noctor and Foyer, 1998). Thus, glutathione may form part of a complex regulatory network underlying adaptation processes and co-ordinating gene expression and cell division. Furthermore, beyond stress perception and signalling processes, glutathione is involved in a wide range of different metabolic functions ranging from detoxification of heavy metals (Cobbett and Goldsbrough, 2002) and conjugation of electrophilic xenobiotics (Marrs, 1996), to reductive processes such as scavenging of reactive oxygen species (ROS) (Noctor and Foyer, 1998). Furthermore GSH is required as a cofactor for several other metabolic processes (Zang et al., 2001; Singla-Pareek et al., 2003) and appears to be essential for root nodule development (Frendo et al., 2005). Glutathione is implicated in the formation of somatic embryos (Earnshaw and Johnson, 1985; Belmonte and Yeung, 2004). It is clear that glutathione is not only important in conferring oxidative stresses but also has key roles in plant development.
Another important group of regulatory molecules are the flavonoids which are found all throughout the plant kingdom. They are characterized by the presence of two benzene rings and are involved in many biological processes such as regulation of pigment synthesis, auxin transport, and pollen germination, as well as signalling to micro-organisms (Koes et al., 2005; Taylor and Grotewold, 2005).
The root and shoot apical meristems (RAM and SAM) are established during embryogenesis and serve as a source of stem cells for plant growth and organogenesis (Scheres et al., 1994). The primary RAM produces all the tissues of the main root by a highly defined pattern of cell divisions in Arabidopsis (Weigel and Jurgens, 2002). Cells produced by the meristem undergo proliferative divisions as they are added to files of different cell types and their fate is determined by positional information (van den Berg et al., 1995, 1998). Stem cells of the root are maintained by the quiescent centre (QC) (van den Berg et al., 1997; Sabatini et al., 2003), which itself is maintained by auxin (Sabatini et al., 2003). Jiang et al. (2003) demonstrated the highly oxidized state of the QC cells in Zea mays and proposed that the QC is established and regulated by ROS generated by auxin. ROS production is thus a downstream component of an auxin-mediated signalling pathway in the root (Joo et al., 2001). Arabidopsis plants homozygous for a mutation in the ROOT MERISTEMLESS1 (RML1) gene which encodes the first enzyme of glutathione biosynthesis, -glutamylcysteine synthetase are unable to establish an active post-embryonic meristem in the root apex. This mutation abolishes cell division in the root but not in the shoot. But the mutants can be rescued by providing seedlings with GSH (Vernoux et al., 2000. The rml1 mutation is a single amino acid substitution (Vernoux et al., 2000). However, the rml1 mutant has detectable residual GSH while another mutant gsh1 (T-DNA insertion) has no detectable residual GSH and confers a recessive embryo lethal phenotype (Cairns et al., 2006). It is clear that glutathione is not only important in response to oxidative stresses but it also has key roles in plant development.
Numerous genes have been identified as specifically expressed during in vitro root formation (Mordhorst et al., 1997; Chugh and Khurana, 2002). These genes include hormone-responsive genes such as those induced by auxin (Walker and Key, 1982) as well as certain homeobox-containing genes (Kawahara et al., 1995; Meijer et al., 1997). It has been shown that WUSCHEL-type homeobox genes, also known as WOX5, are not only expressed in the QC of the RAM in Arabidopsis (Kamiya et al., 2003) but also play a key role in RAM formation during both embryonic and post-embryonic root formation (Haecker et al., 2004; Xu et al., 2006), and it can be induced by auxin (Gonzali et al., 2005). Another group of transcription factors that are involved in root formation are the AP2/EREBP (APETALA2/ethylene responsive element binding family transcription factors) proteins that make up one of the largest transcription factor families. One of the groups that contain two DNA-binding domains is the AP2 subfamily. Data obtained so far for members of this protein family in Arabidopsis, petunia, maize, rice, and tobacco suggest that members of the AP2 subfamily play roles in development (Riechmann and Meyerowitz, 1998). APETALA2, AINTEGUMENTA (ANT), and BABY BOOM (BBM) are shown to be involved in the control of flower and seed development (Bowman et al., 1989; Jofuku et al., 1994; Elliott et al., 1996; Klucher et al., 1996; Boutilier et al., 2002) and in the control of seed size (Elliott et al., 1996; Ohto et al., 2005). One member of the AP2 family that has been implicated in a variety of critical plant cellular functions is the BBM protein. This protein from Arabidopsis is preferentially expressed in seed and has been shown to play a central role in regulating embryo-specific pathways. Overexpression of BBM has been shown to induce spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Two other members of the AP2 subfamily, PLETHORA1 (PLT1) and PLETHORA2 (PLT2), were found to be required for specification and maintenance of stem cells within the RAM, and PLT mRNA distribution is regulated by an auxin maximum that is distal to the vascular precursors (Aida et al., 2004). Recently, it was reported that AINTEGUMENTA-like (AIL) genes are expressed in young tissues and may specify meristematic or division-competent states (Nole-Wilson et al., 2005). Although extensive research has been carried out on root formation, mainly in Arabidopsis, the underlying molecular mechanisms of root induction are not well understood. In particular, no such studies have been reported in legumes. Hence the report here is on factors that effect in vitro root formation in the model legume Medicago truncatula, using quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR), which is estimated to be at least 100-fold more sensitive than DNA arrays in detecting transcripts (Horak and Snyder, 2002).
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. The use of leaf explant tissue culture has the advantage of being a system whereby phytohormones can easily be manipulated to direct pluripotent cells to a particular cell fate (Schmidt et al., 1997; Nolan et al., 2003; Thomas et al., 2004; Imin et al., 2005). When M. truncatula explant tissues are cultured in the presence of high auxin concentrations, they produce numerous roots (Nolan et al., 2003). Earlier histological studies of auxin-induced root formation in leaf explants of M. truncatula showed that there were cells associated with the leaf veins, which can be readily stimulated by adding auxin (Rose et al., 2006). These vein-associated cells were stimulated to divide in response to auxin and grow distinctive sheets of callus cells that emanate from the veins of the leaf explants. These procambial-like cells, which function as pluripotent stem cells can be switched into forming either roots or somatic embryos depending on the prevailing hormone status of the media (Rose et al., 2006). In this study, leaf explant tissues of M. truncatula have been used to investigate the early effect of cytokinin, glutathione, flavonoids, and several key transcription factors including the AINTEGUMENTA-like group in auxin-induced root formation.
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Plant materials, growth, and tissue culture
Medicago truncatula cv. Jemalong seed line 2HA was used for the plant growth explant tissue culture as described (Nolan and Rose, 1998; Nolan et al., 2003). Seeds of M. truncatula cv. Jemalong were obtained from the National Medicago Collection (Northfield Research Laboratories, Adelaide, South Australia). The great advantage of the leaf explant system used in this work is that pieces of leaves can be placed into tissue culture, incubated, and they will form calli and under the appropriate hormone conditions roots or embryos. The sickle (skl) mutant of Jemalong A17 was kindly provided by Professor Doug Cook, UC Davis). This enables the effects of specific plant mutants on a particular developmental pathway to be investigated. Plants were grown under controlled growth cabinet conditions with a 12 h photoperiod at 150 µmol m–2 s–1 with a day temperature of 23 °C, 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 (Thomas et al., 1990). In the usual culture procedure, leaf explants were plated onto P4 medium containing 10 µM 1-naphthaleneacetic acid (NAA; Sigma-Aldrich, St Louis, MO, USA) and/or 4 µM 6-benzylaminopurine (BAP; Sigma-Aldrich). Cultures were incubated in the dark at 28 °C. Both reduced and oxidized forms of glutathione (GSH and GSSG; Sigma-Aldrich) were dissolved in water (10–2 M) and added to the media after autoclaving with the final concentration of 20 mM. All flavonoids (10–2 M) were dissolved in dimethyl sulphoxide (DMSO; Sigma-Aldrich) and added to the media after autoclaving to the final concentration of 10 µM. N-1-Naphthylphthalamic acid (NPA) was dissolved in methanol.
Seeds were scarified, surface-sterilized with 6% hypochlorite solution and washed seven times with sterile distilled water. Seeds were germinated on nitrogen-free Fahraeus medium (Fahraeus, 1957) in Petri dishes in the dark for 24–30 h. They were transferred to new Petri dishes, 14–16 seedlings per plate, and grown for a further 3 d in a growth chamber until the roots had reached a length of 3–4 cm. Plates were kept in a vertical position and the bottom half of each plate was sealed with Nescofilm (NESCO, Japan). Light was kept away from the roots by the insertion of a black sheet between the dishes during incubation. An aluminium foil spacer was placed under the lid of the Petri dish to allow gas exchange. Dishes were incubated in a growth chamber at 25 °C over a 16 h photoperiod at 150 µE m–2 s–1 light intensity with 70% relative humidity. To compare meristematic and non-meristematic root tissues, root sections were harvested from 3-d-old plants. Tissue 3 mm from the root tip which contains the meristematic cells and a further 1 cm section from the root containing non-meristematic differentiating and elongating cells (elongation zone) were collected. All harvested plant materials were immediately frozen in liquid nitrogen and stored at –80 °C.
Histology
Histological and cell biology experiments were done as described elsewhere (Rose et al., 2006).
Cloning and sequencing of PLETHORA-like genes in M. truncatula
All oligonucleotide primers were ordered from Sigma Genosys (Castle Hill, NSW, Australia). One-step 3'-RACE (rapid amplification of cDNA ends) was employed for the amplification of PLETHORA-like genes in M. truncatula. Briefly, total RNA was isolated from 1-week-old 10 µM NAA-treated explant tissue cultures of M. truncatula line 2HA using the Qiagen RNeasy MINI kit (Qiagen, Clifton Hill, Victoria, Australia) according to the manufacturer's instructions. cDNA synthesis was done using 5 µg RNA. RNA was treated in 1x buffer with 2 U of DNase I (Ambion, Austin, TX, USA) added to the reaction and incubated for 30 min at 37 °C. The reaction was stopped by adding DNase removal reagent (Ambion). One microlitre of 5 µM oligo dT18-adaptor primer (5'-GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TTT-3') was added to the reaction, and incubated for 10 min at 70 °C, then chilled on ice. First-strand mix containing 1x buffer, 10 mM DTT, 1.25 mM of each dATP, dCTP, dTTP, and dGTP, was added to a total volume of 20 µl and incubated for 5 min at 42 °C, then 200 U SuperScriptTM III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The reaction was stopped by incubation at 70 °C for 15 min. One microlitre of RNase H (Invitrogen) was added and the mixture incubated at 37 °C for 20 min. The final reaction was either stored at –20 °C or used immediately for PCR.
To identify M. truncatula PLETHORA orthologues, the Arabidopsis PLETHORA sequences (PLT1 and PLT2; GenBank AY506549 and AY506550, respectively) were used for searching nuclei acid databases. Only one PLT orthologue was identified in soybean (TIGR TC205929), Lotus japonicus (GenBank AP007400), and M. truncatula (IMGAG 1162.m00011). Then the PLETHORA genes were aligned using the program MaliN (Softberry Inc., NY, USA) and the forward degenerative primer, PLTconsF (CAACAYGGRAGRTGGCAAGCAAG, where R=A or G; Y=C or T) was designed from the most conserved region. The adaptor primer used was 5'-GAC TCG AGT CGA CAT CGA-3'. PCR was performed using Invitrogen Platinum® Taq DNA polymerase High Fidelity (Invitrogen) according to the manufacturer's instructions using the two primers above, except that the anneal temperature was changed from 55 °C to 58 °C and only 20 cycles were performed. The PCR product was then purified with Qiagen MinElute PCR Purification Kit (Qiagen) according to the manufacturer's instructions using a microcentrifuge.
The TOPO TA cloning kit for sequencing (Invitrogen) combined with the One Shot® Chemical Transformation Protocol (Invitrogen) was used for ligation and to transform into One Shot® TOP10 cells (Invitrogen) according to the manufacturer's instructions. Thirty positive colonies were picked and cultured in LB (Luria–Bertani) medium with kanamycin overnight for analysis. Isolation of plasmid DNA was performed using Qiagen QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer's instructions. Plasmid DNA concentrations were determined by a UV spectrophotometer. Sequencing reaction was carried out in a total volume of 20 µl with 150–300 ng of plasmid DNA, BigDye Terminator v3.0 (Applied Biosystems, CA, USA), 5x buffer, and M13F primer. The thermocycler conditions were as follows: 96 °C for 1 min, then 25 cycles of 96 °C for10 s, 50 °C for 5 s and 60 °C for 4 min. The extension PCR products were purified using ethanol/sodium acetate precipitation procedure and were sequenced on an ABI 3730 capillary genetic analyser (Applied Biosystems) at the Biomolecular Resource Facility (BRF), Australian National University, Australia.
Sequence analysis
Medicago truncatula orthologues of Arabidopsis WOX4 (GenBank AY251396), WOX5 (AY251398), PLT1 (AY506549), PLT2 (AY506550), BBM1 (AF317907), SHOOT MERISTEMLESS (STM) (NM_104916), SCARECROW (SCR) (NM_115282), and SHORT ROOT (SHR) (NM_119928) were identified by using the program BLAST (Altschul et al., 1990) against TIGR gene index (MtGI) and genomic DNA database annotated by IMGAG (International Medicago Genome Annotation Group). The best matches were counter BLAST searched against sequence databanks at the NCBI (Bethesda, USA) to confirm the matches. Cloned gene sequences were recovered by homology search in sequence databanks using the BLAST at the NCBI or at TIGR.
Quantitative real-time RT-PCR
Total RNAs were isolated from tissues of M. truncatula line 2HA using the Qiagen RNeasy MINI kit (Qiagen). Total RNA was treated with DNase I (Ambion) and inactivated before the first strand cDNA synthesis. cDNA synthesis was same as above except that 2 µg total RNA for each sample was used and oligo dT18 (5'-TTT TTT TTT TTT TTT TTT-3') was used instead of adaptor dT18 oligo. For the no reverse transcriptase control, water was added instead of SuperScript III reverse transcriptase. For the real-time RT-PCR, gene-specific primers (Table 1) were designed using Primer Express software (Applied Biosystems) and ordered from Sigma Genosys. The PCR was carried out in a total volume of 20 µl containing 0.2 µM of each primer, 1x SYBR green PCR master mix (PE Applied Biosystems). Reactions were amplified as follows: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, 60 °C for 1.5 min. Amplifications were performed in 0.2 µl MicroAmp optical tubes (PerkinElmer, Wellesley, MA, USA) with an ABI PRISM 7700 sequence detection system (at the Biomolecular Resource Facility, JCSMR, ANU) using version 1.9 software (Applied Biosystems) to analyse raw data. The absence of genomic DNA and non-specific by-products of the PCR amplification was confirmed by analysis of dissociation curves and agarose gel electrophoresis of the PCR products. Data were analysed using the SDS 1.9.1 software. Normalization was done as described (Searle et al., 2003) against the MtUBQ10 (ubiqutin 10, TC100142) gene by calculating differences between the CT of the target gene and the CT of ubiquitin 10, Relative gene expression levels were calculated by plotting against the lowest expressed sample after the normalization. Three biological repeats (independent tissue-culture experiments performed in parallel under the same growth conditions) were done for each treatment. For each sample, a no RT enzyme reaction was also performed in triplicate. The PCR products were also electrophoresed in 3% agarose gels. The gels were stained with 0.5 µg ml–1 ethidium bromide, visualized using a UV transilluminator, and then photographed.
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Cytokinin inhibits in vitro root formation and auxin pretreatment interrupts embryo formation
In M. truncatula, exogenous application of auxin such as NAA to the leaf explants can induce root-like structures but not embryos (Fig. 1A). When both auxin and cytokinin such as BAP were applied, the superembryogenic line 2HA produces embryo-like structures but no root-like structures (Fig. 1B), although a limited number of embryos could form on cytokinin alone (Fig. 1C). The earlier experiments of Schmidt et al. (1997) and Thomas et al. (2004) on carrot and sunflower tissues showed that there was an irreversible step that occurs during the initial exposure of the inducing hormones. Therefore a test was carried out to determine if the same phenomenon took place in M. truncatula tissues using the embryogenic line 2HA, which can be triggered to form roots on auxin or somatic embryos on auxin plus cytokinin media (Fig. 1). Clearly, after exposure for 1 week to auxin, the commitment to rooting cannot be reversed by shifting to the embryogenic medium (auxin plus cytokinin). Figure 1D–F shows the effect of cytokinin addition after initial exposure to auxin for 1, 2, and 4 weeks, respectively. After exposure for 1 week to auxin-containing medium the cultures form fewer roots and no embryos on the auxin- and cytokinin-containing medium (Fig. 1F). The longer the exposure to auxin the fewer the roots formed, but no embryo formation was observed. Leaf explants placed into auxin plus cytokinin medium from the start formed somatic embryos (Fig. 1B). Thus, a pattern of commitment and differentiation is established by the first exposure and once set up it is not easily reversed.
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Glutathione enhances while flavonoids inhibit in vitro root formation
Using leaf explants cultured on medium containing auxin (NAA) it was possible to markedly enhance the number of roots formed from each explant callus by the addition of either the reduced (GSH) or oxidized (GSSG) form of glutathione (Figs 2, 3, columns 3, 4). The skl mutant has a defect in ethylene perception, is ethylene-insensitive, and has been mapped and sequenced and shown to be an orthologue of the EIN2 gene of Arabidopsis which is a nuclear membrane protein involved in the transmission of the ethylene signalling pathway (Penmetsa and Cook, 1997; D Cook, personal communication). The increase in rooting was also observed with the ethylene-insensitive mutant skl cultures after the addition of glutathione (Fig. 3, columns 5, 6). Early studies showed an increase in root number with the ethylene-insensitive mutant skl cultures when compared with wild-type Jemalong A17 (Rose et al., 2006). Here it was observed that upon glutathione addition to the skl mutant cultures there was a synergistic effect on the number of roots formed per callus piece (Fig. 3, columns 5, 6).
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A range of flavonoid molecules was added to root-forming leaf explant cultures to test their influence on root formation (Fig. 4). The isoflavones, formononetin and genistein, had the biggest effect on the onset of rooting, the growth rate, and the number of roots formed per callus. Quercetin, which may inhibit polar auxin transport, also influenced root growth but to a lesser degree. NPA, an auxin transport inhibitor that resembles quercetin, was also shown to reducte root growth and root numbers. The other flavonoids tested, naringenin, luteolin, apigenin, and dihydroxyflavone, all had an effect on reducing root growth and root numbers compared with the control cultures, but to a lesser extent compared with the isoflavones.
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Gene expression during in vitro root formation
Several transcription factors that are well known for their role in Arabidopsis root formation have been chosen (Kamiya et al., 2003; Aida et al., 2004; Haecker et al., 2004; Nole-Wilson et al., 2005; Xu et al., 2006). The WUSCHEL-related homeobox WOX5 and AP2-domain containing transcription factors PLETHORA (PLT1 and PLT2) were used to examine their transcript expression during the initiation of commitment and differentiation in the leaf explant tissues of M. truncatula (Table 1). First M. truncatula orthologues of Arabidopsis genes such as WOX4, WOX5, PLT2, BBM1, SCR, and SHR were identified by searching against TIGR Medicago gene index and genomic DNA database. Identified sequences were searched again against the Arabidopsis database to confirm the orthologues. The orthologues accorded were defined using the criteria below: Gene A in species X should identify gene B in species Y as the highest ranking match by BLAST search and gene B in species Y should also identify gene A in species X as the highest ranking match. All of the matches had higher than 50% identities and the expected values were lower than e–30. Quantitative real-time PCR was performed on leaf explant tissue sampled at the initiation of commitment. One-week-old explant cultures were chosen to identify genes involved in the early auxin-induced root-formation process. It should be noted that root-like structures start to appear after 2 weeks of culture when treated with exogenous auxin. At 1 week no roots have appeared; however, micro-calli have started to emerge from the edge of the explant leaf. It is from this tissue that root primordia will eventually form. Prior to that, there is no visible morphological change that can be observed (data not shown).
As shown in Fig. 5, MtWOX5 was highly up-regulated (997-fold increase compared with leaf) in 1-week-old root-forming calli. However, when both auxin and cytokinin were applied, the change was reduced to 348-fold (35% decrease). Also, a low level of MtWOX5 expression in root tips (57-fold increase compared with that of the root elongation zone) has been observed. The gene MtPLT2 was highly expressed in root tips (83-fold compared with that of the elongation zone) and was induced in 1-week-old root-forming calli (417-fold compared with that of 1-week-old embryogenic calli). Cytokinin addition appeared to inhibit MtPLT2 expression completely (Fig. 5). Also, the involvement of the well-known early embryo-expressed AP2 domain-containing transcription factor BBM (Boutilier et al., 2002) has been studied in root formation. Interestingly, the M. truncatula orthologue MtBBM1 responded extremely well to auxin addition with a 1408-fold increase in 1-week-old root-forming calli grown on 10 µM NAA compared with that of leaf. However, its expression was also inhibited by cytokinin addition (reduced to 240-fold which is an 83% reduction compared with that of 1-week-old root-forming calli). Moreover, an enrichment of MtBBM1 has been observed in the root tip, but not in the elongation zone (Fig. 6).
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To identify PLETHORA orthologues in M. truncatula, first Arabidopsis PLT genes were used to search for orthologues in other species. One was found from each species of soybean, Lotus japonicus, and M. truncatula. However, due to the incomplete sequence in M. truncatula, it was not possible to identify other potential orthologues of PLT genes. All of these PLT genes were then aligned and a conserved degenerative primer which was used in one-step 3'-RACE and gene cloning was designed. By screening 30 positive colonies and sequencing, three positive colonies with inserts that all belong to the AINTEGUMENTA-like group have been identified. After BLAST searches, they were identified as orthologues of PLT1, ANT (AINTEGUMENTA), and AINTEGUMENTA-like (AIL) and as such were named MtPLT1, MtANT, and MtAIL1, respectively (Table 1). Of these three genes, MtPLT1 showed an extremely high level of induction by exogenous application of auxin. For instance, 1-week-old root-forming calli grown on 10 µM NAA had a 14 450-fold increase in gene expression compared with that of the leaf. Similar to MtPLT2 expression, MtPLT1 was also inhibited by the additional cytokinin. However, this inhibition was much more dramatic. For instance, in 1-week-old embryogenic calli grown on 10 µM NAA and 4 µM BAP, MtPLT1 had a 99.5% decrease in gene expression compared with that of 1-week-old root-forming calli. Furthermore, like MtPLT2, the expression of MtPLT1 was also enriched in root tips (9476-fold compared with that of the elongation zone). Both MtANT and MtAIL1 had some slight increase (51- and 3.5-fold increase, respectively compared with that of the leaf) in 1-week-old root-forming calli grown on 10 µM NAA. By contrast to MtBBM1, MtPLT1, and MtPLT2, cytokinin addition elevated the expression of MtANT and MtAIL1 instead of having an inhibitory effect. In the case of MtANT, there was no enrichment in root tips compared with the elongation zone, although there appeared to be some increase (30-fold) in MtAIL1 in the root tips compared with the elongation zone. However, the overall changes in MtANT and MtAIL1 expression were not as dramatic as for MtWOX5, MtBBM1, MtPLT1, and MtPLT2.
Also the relative expression of GRAS family transcription factors SCR1 and SHR1 that are involved in the maintenance of the root QC have been measured. Other transcription factors examined included SHOOT MERISTEMLESS (STM, also known as KNOX1) which is required for SAM formation (Long et al., 1996) and WOX4 which is specifically expressed in procambial cells (J Ji and M Scanlon, personal communication). While MtSCR1 had slightly higher expression in all the samples tested when compared with leaf tissue, MtSHR1 transcript was mainly enriched in root tips with a slight expression detected in the elongation zone. MtSHR1 transcript did not appear to be induced by auxin and/or cytokinin, while MtSCR1 had some slight induction by auxin and/or cytokinin addition. These results were consistent with the microarray analysis of similar samples in M. truncatula (N Imin et al., unpublished data). As expected, auxin addition did not induce expression of MtSTM and MtWOX4 by contrast to transcription factors known to be involved in root formation, such as MtWOX5, MtPLT1, and MtPLT2 or MtBBM1. However, cytokinin addition can induce the transcription factors MtSTM and MtWOX4 dramatically. A 97-fold and 50-fold increase in MtSTM and MtWOX4 expression was observed, respectively, in 1-week-old embryogenic calli grown on 10 µM NAA and 4 µM BAP (compared with that of the leaf). Some low level expression of MtSTM and MtWOX4 was observed in root tips as well as the root elongation zone.
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When M. truncatula leaf explant tissues are cultured in medium containing auxin, they produce numerous roots (Nolan et al., 2003). Earlier histological studies of auxin-induced root formation in leaf explants of M. truncatula showed that there were cells associated with the leaf veins, which can be readily stimulated to form roots by the exogenous application of auxin (Rose et al., 2006). These vein-associated cells were stimulated to divide in response to auxin and grow distinctive sheets of callus cells that emanate from the veins of the leaf explants. They are procambial-like cells, which function as pluripotent stem cells that can be switched into forming either roots or somatic embryos depending on the prevailing hormone medium conditions (Rose et al., 2006). Leaf explant tissues of M. truncatula have been used to investigate the effect of auxin, cytokinin, glutathione, flavonoids, and several key transcription factors in auxin-induced root formation on the commitment process to differentiation.
Factors that influence in vitro root formation
During the initial phases of organogenesis, somatic cells progress through a series of events referred to as differentiation, competence acquisition, induction, and determination (Thomas et al., 2004). 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 (Dudits et al., 1991; Thomas et al., 2004). Working with immature zygotic embryos of sunflowers, Thomas et al. (2004) 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 (Thomas et al., 2004). The period lasted for 3 d when the commitment could be reversed by changing the medium. However, after 4 d it could not be altered and thus an irreversible step was taken within the competent cells toward a particular organogenesis pathway. The experiments with M. truncatula leaf explants have also revealed an irreversible stage of commitment to either root formation or somatic embryogenesis. If the exposure to auxin is 7 d or more before shifting the tissues onto auxin plus cytokinin medium then embryogenesis will not occur. Auxin induces and cytokinin inhibits root formation, and the longer the exposure time to auxin the less likely the cytokinin inhibition of root formation will take place. Furthermore, the competent cells that are committed to root formation do not reverse to form embryos even when auxin and cytokinin are supplied at the appropriate concentrations. The cells committed to become roots by 7 d cannot form somatic embryos in M. truncatula leaf explants, thus indicating that an irreversible step has been taken within the competent cells toward the root organogenesis pathway.
Earlier observations (Rose et al., 2006) suggested that a pool of stem cells exists in the vascular tissue and that a combination of auxin and other factors drive cellular commitment and plant development. The present studies show that some of these other factors, which specially influence root formation, include the redox environment as indicated by the redox state of glutathione, specific flavonoids, auxin, ethylene, and cytokinin. Rose et al. (2006) showed that when leaf explants of the ethylene-insensitive mutant skl were placed into culture then there was a significant increase in rooting. The present results show that adding either GSH or GSSG molecules to the medium could further enhance the degree of rooting. This indicates that the ethylene and redox conditions play an important role in the initial commitment step of root formation and glutathione enhances root formation via a mechanism independent of ethylene perception. Jiang et al. (2003) demonstrated the highly oxidized state of the QC cells in Zea mays. This highly oxidized state of the QC was determined via the presence of oxidized forms of both glutathione and ascorbic acid. The authors proposed that the QC of the RAM is established and regulated by the ROS generated by auxin. The redox status of the QC is different from that of the adjacent rapidly dividing cells and Jiang and Feldman (2005) proposed that auxin affects the cell cycle in the QC via changes in redox. ROS production is thus a downstream component of an auxin-mediated signalling pathway in the root (Joo et al., 2001) and is most probably involved in maintaining a slow rate of cell division within the QC. Perhaps the inhibitory controller of daughter cell differentiation is in the QC redox. The initial exposure to auxin therefore rapidly affects the decision-making process to commitment within the vein-derived cells that cannot be reversed and therefore auxin triggers the expression of key transcription factors that mediate the formation of the root primordium before the QC is formed (Rose et al., 2006). Although this auxin-induced commitment to rooting is irreversible, it can be influenced, in that the rate of onset and growth of the induced roots formed can be reduced by the addition of different flavonoid compounds. It is interesting to note that, of the seven flavonoid compounds tested, strongest inhibition of rooting was observed for formononetin and genistein, which belong to the legume-abundant isoflavone subfamily (Paiva et al., 1994; Hosny and Rosazza, 1999). Given the abundance of these isoflavones in legumes it may be worthwhile to test other members of this family on root formation. Quercetin, the third strongest flavonoid inhibitor of in vitro root formation tested, has been reported to have a similar structure to NPA (Murphy et al., 2000) which also showed in vitro root inhibition in M. truncatula. NPA has been shown to interfere with the intracellular cycling of the PIN proteins between the plasma membrane and endosomal vesicles in the presence of brefeldin A, an inhibitor of endosomal transport (Geldner et al., 2001). As such, quercetin may function as an auxin transport inhibitor. Inhibition of auxin transport may prevent specific cell populations from reaching a localized auxin maximum which is required for the establishment of root primordia.
Genes that mediate in vitro root formation
To analyse these initial steps of commitment further, a series of quantitative real-time PCR experiments examined the expression of the several transcription factors. They included two homeobox WUSCHEL-like genes WOX4 and WOX5, five AP2-domain-containing AINTEGUMENTA-like genes (PLT1, PLT2, ANT, AIL, and BBM1), two GRAS family transcription factors, SCR and SHR, and class I KNOX gene, STM. WOX5, SCR, SHR, PLT1, and PLT2 are known to be associated with the formation of the RAM and QC in Arabidopsis (Helariutta et al., 2000; Sabatini et al., 2003; Aida et al., 2004; Haecker et al., 2004; Xu et al., 2006). In Arabidopsis, WOX5, PLT1, and PLT2 are reported to be expressed in the QC and induced by auxin (Aida et al., 2004; Xu et al., 2006), linking auxin signalling with root formation. The present data showed that the genes MtWOX5, MtPLT1, MtPLT2, and MtBBM1 are extremely responsive to auxin presence and were found to be highly expressed in 1-week-old root-forming calli. As described in Results, root-like structures appear only after 2 weeks of culture when they are supplied with exogenous auxin. At 1 week, there is no root formation, although micro-calli appear at the margins of the explant leaves. Ultimately root primordia form from these micro-calli. Furthermore, the root-tip that contains the QC was also enriched with all of the above genes, confirming the importance of these transcription factors in the development of the RAM and indicating similarity between Arabidopsis and the legume plant M. truncatula. Expression of these genes as early as 1 week indicates a role for them not only in the formation and maintenance of the QC within roots but also in the formation of niches for root primordia that precede the QC formation. This is consistent with their inhibition by exogenous cytokinin addition, which enables the production of somatic embryos but inhibits rooting. By contrast, the gene STM, which is known to be induced by cytokinin and is involved in the SAM (Long et al., 1996; Jasinski et al., 2005) was clearly expressed on auxin plus cytokinin medium. Similar results were also obtained for WOX4 which was shown to be involved in vascular primordia formation in tomato (J Ji and M Scanlon, personal communication). Both the ANT and AIL genes were stimulated by the presence of auxin and cytokinin in the medium and weakly expressed in the root tip and elongation zone. It is concluded that these genes are less important to the initial steps in the commitment stage of root development. BBM is an embryo-expressed transcription factor that plays a key role in the initiation and maintenance of embryo development in Arabidopsis (Boutilier et al., 2002). A recent study also showed that BBM along with other AINTEGUMENTA-like genes are expressed in young tissues including roots, and may specify meristematic or division-competent states (Nole-Wilson et al., 2005). For the first time, these results show that the BBM transcript was enriched in root tips which would suggest a role for BBM in auxin-mediated M. truncatula root primordia formation.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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There are a specific group of cells (probably procambium-like cells) in the leaf explants which are sensitive to prevailing phytohormone balances, and whose changes in this balance can induce specific steps in commitment to different differentiation pathways. Auxin helps to lock in the steps to root and root vascular development in a way that is not easily reversed. Thus the question of the plasticity of plant development may reflect which hormone-inducing system was present when cells were first tested. Furthermore, this commitment to rooting can be greatly enhanced by the addition of several exogenous agents such as redox compounds including glutathione. This idea of plasticity is important to the whole area of adult cell reprogramming, de-differentiation, and pluripotency. Stem cell biology in plants and animal systems is still a young field of research but it is fundamental to basic biological systems.
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Acknowledgements |
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We acknowledge Chris Dawson and Nikki Schultz for their contributions to the project and we thank Jeff Wilson for the photography, and Peta Holmes and Michael Djordjevic for their valuable suggestions. This work was supported by the Australian Research Council (Grant no. CEO348212).
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Abbreviations |
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AIL, AINTEGUMENTA-like; ANT, AINTEGUMENTA; BAP, 6-benzylaminopurine; NPA, N-1-naphthylphthalamic acid; BBM, baby boom; DMSO, dimethyl sulphoxide; GSH, glutathione (reduced form); GSSG, glutathione (oxidized form); NAA, 1-naphthaleneacetic acid; PLT, PLETHORA; QC, quiescent centre; RACE, rapid amplification of cDNA ends; RAM, root apical meristem; ROS, reactive oxygen species; RT-PCR, reverse transcription–polymerase chain reaction; SAM, shoot apical meristem; SCR, SCARECROW; SHR, SHORT ROOT; skl, sickle; STM, SHOOT MERISTEMLESS.
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