Introduction
Assembly of a patterned multicellular organism from isolated pieces of adult somatic tissues occurs in both animals and plants (
Morgan, 1901). But it is rare for a complete organism regenerated from cells of somatic tissues in animals, except from the embryonic stem cells (
Evans and Kaufman, 1981;
Thomson et al., 1998). Unlike animal cells, plant cells have a profound capacity to regenerate new tissues, organs, and the whole body when properly cultured because they have been thought to maintain totipotency (
Morgan, 1901;
Birnbaum and Sánchez Alvarado, 2008). Although plant cells can differentiate to regenerate various plant organs, stem cells remain undifferentiated in the meristem to maintain their ability to divide and generate new cells for sustained growth. Plants maintain a balance of the cell fate in meristems, where the input of dividing pluripotent stem cells offsets the output of differentiating cells (
Gross-Hardt and Laux, 2003;
Rieu and Laux, 2009). Two stem cell populations arise during embryogenesis and grow postembryonically in a polar fashion throughout the plant life. The shoot apical meristem (SAM) generates the aerial part of the plant, whereas the root apical meristem (RAM) gives rise to the underground part (
Jürgens, 2001;
Gross-Hardt and Laux, 2003). However, there have been relatively few reports on the pattern formation of stem cells during the
de novo assembly of the Arabidopsis meristems.
The expression of
WUSCHEL (
WUS) and
CLAVATA3 (
CLV3) is required for the establishment and maintenance of embryonic SAM (
Clark et al., 1996;
Laux et al., 1996;
Long et al., 1996). WUS homeodomain transcription factor maintains neighboring stem cells in the shoot organizing center (OC), and ectopic expression of
WUS is sufficient to stimulate SAM regeneration (
Zuo et al., 2002;
Gallois et al., 2004).
CLV3 gene is expressed in the superficial layers of those SAM central cells that are considered to be stem cells, where it is activated by WUS (
Fletcher and Meyerowitz, 2000). The CLV complex (CLV3 and CLV1) regulates the size of the meristem by restricting WUS action in turn (
Schoof et al., 2000).
In the RAM, the common regulatory mechanism for stem cell maintenance is involved in a
WUS homolog,
WUSCHEL RELATED HOMEOBOX5 (
WOX5).
WOX5 expression marks the quiescent center (QC), where its primary function is to maintain distal stem cells (
Sarkar et al., 2007;
Ding and Friml, 2010). The alternative feature of
WOX5 is that its proteins prevent the surrounding stem cells to differentiate as a postulated short-range factor (
van den Berg et al., 1997;
Sarkar et al., 2007). The other important regulators of the root stem cell activity include PLETHORA (PLT) genes, a few AP2-domain transcription factors (
Aida et al., 2004;
Galinha et al., 2007). The
plt1 plt2 plt3 triple mutant shows the rootless phenotype. In contrast, constitutive expression of
PLT2 can induce ectopic root formation, which causes the increase in stem cell number and activity (
Galinha et al., 2007). Thus, the transcriptional level of
PLT is very important to the determination of stem cell fate (
Aida et al., 2004;
Galinha et al., 2007;
Terpstra and Heidstra, 2009).
In this study, we conducted a detailed examination of the stem cell differentiation during different organ regeneration in Arabidopsis. We used a set of marker lines to monitor the stem cell specification. Our results show that the regeneration of meristem in callus is due to re-determination of cell fate or the formation of stem cells. Stem cells in callus exhibit their totipotency to differentiate into different types of meristems under different cultured conditions. Different patterns of stem cell fromation suggest that regulation of stem cell differentiation might be responsible for the determination of organ types.
Materials and methods
Plant materials
The Arabidopsis thaliana plants used in this study were Columbia and Wassilewskija ecotypes. The pWUS::DsRED-N7 pCLV3::GFP double marker line was obtained from Dr. E. M. MEYEROWITZ (Division of Biology, California Institute of Technology, USA) in the Columbia ecotype. The pWOX5::GFP-ER seeds were kindly provided by Dr. J. XU (Department of Molecular Genetics, Utrecht University, The Netherlands).
Growth and cultured conditions
Arabidopsis surface-sterilized seeds were germinated on MS medium (
Murashige and Skoog, 1962). Post-germinated seedlings were cultured at 22°C with a 16 h light/8 h dark cycle, and then transferred to conical flask under the same condition. Pistils as explants were cultured in callus induced medium (CIM) that contains 0.5 mg/L 2,4-D with 1.0 mg/L 6-BA for callus induction according to Cheng et al. (
2010). Then, the calli were transferred to shoot induction medium (SIM) that contains 0.01 mg/L indole-3-acetic acid (IAA) and 2.0mg/L zeatin (ZT). (indole-3-acetic acid) for shoot induction. In separate experiments, the calli on CIM for 20 days were transferred to root induction mediem (RIM) that contains 0.01 mg/L IAA for root regeneration.
Plasmid construction
To construct
pPLT2::RFP, the 2.6 kb-
PLT2 promoter region was PCR-amplified using the primers PLT2-1 (5′-CAGTTGATCGCTGTTAGAC-3′) and PLT2-2 (5′-AACGCAAGTTTGGTAAAGA-3′). The PCR products were cloned into the pBI121 expression vector. The construction of
pWUS::GUS had been described previously (
Su et al., 2009).
GUS staining
GUS activity was detected as described by Sieburth and Meyerowitz (
1997). Tissues were stained in the GUS assay solution at 37°C for 12 h. The stained tissues were then fixed in 70% ethanol and embedded in paraffin (Sigma).
In situ hybridization
In situ hybridization was performed according to standard protocols by Zhao et al. (
2005). For
in situ hybridization, calli were fixed in formalin/acetic acid/alcohol (FAA) overnight at 4°C. After dehydration, the fixed tissue was embedded in paraplast (Sigma) and sectioned at 8 μm thickness. The primers to make the templates for
WUS transcripts are WUS-F (5′-GTACTAGTGCCACAGCATCAGCATCATCAT-3′) and WUS-R (5′-GTCTCGAGCTAGTTCAGACGTAGCTCAAGAG-3′). The RNA probes were synthesized using a digoxigenin RNA labeling kit (Roche, Germany).
Imaging acquisition
The callus tissues, somatic embryos, and regenerated shoots and roots were photographed with an Olympus JM 180 dissecting microscope. Embryonic calli were photographed using a scanning electron microscope as described by Li et al. (
2002). For confocal microscopy images, the calli from
pWUS::DsRED-N7 pCLV3::GFP double marker line,
pWOX5::GFP-ER and
pPLT2::RFP marker lines were examined using a Zeiss 510 Meta laser scanning confocal microscope with 20× air and 40× oil objective lenses. Sets of filters used to visualize green fluorescent protein (GFP) and red fluorescent protein (RFP) were selected as described previously by Su et al. (
2009). Zeiss LSM software was used to analyze the confocal imaging.
Results and discussion
Induction of stem cells during somatic embryogenesis
Previous studies indicate that somatic embryogenesis is morphologically and physiologically similar to zygotic embryogenesis in many respects in carrots and
Arabidopsis (
Higashi et al., 1998;
Shiota et al., 1998,
1999). To obtain sufficient numbers of somatic embryos, we established an improved system for somatic embryogenesis in
Arabidopsis. Several green primary somatic embryos were directly regenerated from zygotic embryo explants. These somatic embryos were then cultured in liquid medium containing 2,4-D (embryonic callus-inducing medium, ECIM) to induce the embryonic callus (Fig. 1A). The embryonic calli were transferred to 2,4-D-free liquid medium (somatic embryo-inducing medium, SEIM) for the induction of the secondary somatic embryos. Somatic embryos (SEs) with cotyledon primordia were visible after 4 days of culture (Fig.1B). Then, a large number of mature SEs with hypocotyls, cotyledons and radicles were observed at day 8 (Fig. 1C).
To further investigate the organization of SAM during somatic embryogenesis, we used both marker lines and in situ hybridization to follow the expression patterns of cell type markers that are expressed in SAM. The pWUS::GUS signals were weakly detected in the central regions of callus in ECIM for 14 days, but there were no signals near the edges of the callus, where the somatic embryos would be derived (data not shown). Then, WUS signals were induced in a group of cells under the outmost 3–4 layers of cells in the edge of embryonic callus at around 24 h after the removal of auxin (Fig. 1D). Each group of cells may represent an OC in the calli. At later stages of somatic embryogenesis, WUS signals were observed in the SAM OC cells in the somatic embryos (Fig. 1E and F). In co-localization analysis, CLV3 expression was first detected in the edges of callus in SEIM at day 2, and was slightly later than WUS expression (Fig. 1G and H). Thereafter, both stem cells and OC cells were localized in the SAM of the SEs (Fig. 1I). Therefore, WUS and CLV3 play key roles in the initiation of SAM and promote somatic embryogenesis through the regulation of stem cell formation. It is likely that the early morphological identity in the embryonic callus is the differentiation of an OC following an inducible treatment, and three layers of stem cells were then generated over the OC.
Specification of stem cells during in vitro shoot regeneration
In plant, a seedling starts with two primary meristems, shoot and root meristems. Both meristems are formed early during embryogenesis, and produce all tissues of shoots and roots, respectively, during the postembryonic development. Shoots and roots can be directly regenerated from the callus, and an
in vitro shoot regeneration system using stage-10 pistils as explants was established in our previous studies (
Cheng et al., 2010). The calli were first induced on the callus induction medium (CIM) that contains 0.5 mg/L 2, 4-D with 1.0 mg/L 6-BA for 20 days (Fig. 2A). When the calli were transferred to the shoot induction medium (SIM) for 6 days, light green tissues emerged from the callus (Fig. 2B), and give rise to young shoots subsequently on SIM at around day 20 (Fig. 2C).
Since the WUS gene plays an critical role in the maintenance of stem cells in the center of the shoot meristem, we determined the pattern of WUS/CLV3 expression during shoot regeneration with pWUS::GUS and pWUS::DsRED-N7/ pCLV3::GFP. marker lines. The signals of WUS expression were not detected in the callus on CIM for 20 days (Fig. 2D). In contrast, its expression was detected under the outmost 5–6 layers of cells in the callus after being transferred to SIM for 4 days (Fig. 2G). The WUS signals were then detected in the initial shoot meristems after 6 days and underneath the outmost three cell layers in the shoot meristems on SIM for 8 days (Fig.2EandF). The expression of CLV3, a stem cell marker, came later than that of WUS. At day 5, CLV3 signals were firstly detected and overlaid the regions of WUS expression (Fig. 2H and I). Our results clearly indicate a spatial-temporal pattern of the OC and the stem cell formation during shoot regeneration. Therefore, WUS/CLV3 expression is developmentally regulated during shoot induction. Similar to the somatic embryogenesis, the induction of the OC preceded that of stem cells, and the OC was located below the region of stem cells during shoot regeneration.
Determination of stem cells during root regeneration
We also examined root meristem regeneration. Following the induction of calli formation from pistil explants on CIM for 20 days (Fig. 3A and B), the calli were induced for root production by a transfer to auxin-rich root induction medium (RIM). After 15 days, young roots were regenerated from the callus (Fig. 3C). The molecular events of the QC and stem cell differentiation were further monitored in both
pWOX5::GFP-ER and
pPLT2::RFP marker lines. Interestingly, we found that the meristem-specific
WOX5 and
PLT2 genes were expressed in the subepidermal layer of cells in callus tissues on CIM. Their expression seems to overlap significantly (Fig. 3D and G). Furthermore, the expression of
WOX5 as well as
PLT2 appeared to be in some specific regions of the callus after culture on RIM for 3 days (Fig. 3E and H). When root primordia was observed at around day 5 on RIM, both
WOX5 and
PLT2 signals were localized in the root meristems of the regenerated roots, just like that of regular plant roots (Fig. 3F and I) (
Xu et al., 2006). These results imply that the differentiation of QC cells and stem cells is developmentally regulated during root regeneration, and the formation of stem cells is closely related to the formation of QC cells.
A model for stem cell formation during various in vitro organogenesis
In this study, we showed that somatic embryos, shoots and roots could be induced from callus tissue under various conditions. Through the analysis of stem-cell specific gene expression, we found that the induction of stem cells and the subsequent establishment of meristem are essential for organ regeneration
in vitro. During the induction of all three types of organs, portion of the cells in the callus must be reprogrammed, and their fates are specified to develop into stem cells. The formation of OC in SAM or QC in RAM precedes the formation of stem cells (Fig. 4), consistent with that previously observed in plant (
Weigel and Jürgens, 2002;
Müller and Sheen, 2008). However, the formation of stem cell during
in vitro organogenesis differs from each other in a few important aspects. First, during somatic embryogenesis,
WUS transcription was detected in the central regions of embryonic calli from embryo explants, but not in the calli directly derived from pistil explants. Therefore, the two types of callus could be fundamentally different in their developmental status, which might be critical for fate determination of stem cells and organ types. Second, the signals of
WOX5 and
PLT2 are specifically expressed along the edge of the callus on CIM during root regeneration; by contrast, the signals of
WUS and
CLV3 are not detected in the callus under non-induced conditions. Last, the time courses of stem cell formation are different during the three regeneration processes for organ induction. The induction of regional expression of
WOX5 on RIM (3 days) is earlier than that of
WUS on SIM (4 days) (Fig. 4), suggesting that the induction of roots is simpler than that of shoots using the pistil explants on the two different inducible media. During the somatic embryogenesis, the expression of
WUS even comes much earlier in the SEIM at about 24 h (Fig.4). These results suggest that stem cell specification is temporally regulated by both the types of explants and their responses to plant hormones.
Organ regeneration between plants and animals
Comparison of tissue or organ regeneration in plants and animals can be framed around their capacity for stem cell renewal. Stem cells self-renew and give rise to all differentiated cell types of the adult body (
Scheres, 2007). Plants and animals are quite similar that they can de-differentiate their partially differentiated cells to generate stem cells. For example, when we excise the root tips or use laser-induced wounding to disrupt the root development, plants can restore their root meristems to form new root tissues (
Prantl, 1874;
Xu et al., 2006). A recent study has demonstrated that the spermatagonia can regain totipotency in
Drosophila testis (
Brawley and Matunis, 2004). Although animal stem cells underwent a one-way road from being totipotent or pluripotent to permanently entering a differentiation pathway according to the traditional view, some types of animal cells can revert to the stem cell fate under special conditions (
Birnbaum and Sánchez Alvarado, 2008). In contrast to animal cells, almost all plant cells are extraordinarily totipotent to regenerate all kinds of tissues and organs. Somatic embryogenesis can be induced not only from young embryos, but also from adult tissues, such as leaves (
Lotan et al., 1998;
Stone et al., 2001). Any organs including roots and shoots could be
in vitro regenerated from roots, stems, leaves and pistils in
Arabidopsis (data not shown). In animals, it seems that only the embryonic stem cells have such totipotency (
Turnpenny et al., 2006;
Birnbaum and Sánchez Alvarado, 2008). We still know little about the molecular basis between plants and animals in their organ regeneration. Understanding how the plant cells reverse their differentiating state to stem cell state might provide important information on the mechanism of stem cell determination in plants as well as in animals.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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