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First published online June 30, 2006; 10.1104/pp.106.083022 Plant Physiology 141:1338-1348 (2006) © 2006 American Society of Plant Biologists Induction of Differentiation in the Shoot Apical Meristem by Transient Overexpression of a Retinoblastoma-Related Protein1Institute of Plant Sciences, Swiss Federal Institute of Technology Zürich, CH–8092 Zurich, Switzerland (J.W., S.P., W.G., A.J.F.); Department of Biology-Plant Biology, University of Fribourg, CH–1700 Fribourg, Switzerland (M.S.); and Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom (A.J.F.)
The shoot apical meristem contains cells that undergo continual growth and division to generate the building blocks for the aerial portion of the plant. As cells leave the meristem, they undergo differentiation to form specific cell types. Most notably, heterotrophic cells of the meristem rapidly gain autotrophic capability by synthesis and assembly of components of the chloroplast. At the same time, cells undergo enlargement via vacuolation. Despite significant advances in the characterization of transcriptional networks involved in meristem maintenance and leaf determination, our understanding of the actual mechanism of meristem cell differentiation remains very limited. Using a microinduction technique, we show that local, transient overexpression of a retinoblastoma-related (RBR) protein in the shoot apical meristem is sufficient to trigger cells in the meristem to undergo the initial stages of differentiation. Taken together with recent data showing that RBR protein plays a key role in restricting stem cell differentiation in the root apical meristem, our data contribute to an emerging picture of RBR proteins as a central part of the mechanism controlling meristem cell differentiation.
The shoot apical meristem (SAM) consists of a group of proliferating cells whose progeny provide the building blocks for all aerial organs of the plant. The continual generation of new cells does not, however, normally lead to a continual increase in the size of the SAM. Instead, cells toward the proximal base of the SAM differentiate and become incorporated into the plant stem, whereas groups of cells on the meristem flank become incorporated at regular intervals in time and space into new organs, the leaves (for review, see Tsiantis and Hay, 2003  ; Byrne, 2005; Fleming, 2005). This process of meristem cell differentiation is associated with and, indeed, in many ways, defined by specific changes in cytology. Thus, whereas cells in the SAM are distinguished by being relatively small and cytoplasmically dense, as cells become incorporated into the stem and leaf structures they become larger as a result of vacuole enlargement, with the cytoplasm being pushed into a layer around the periphery. Simultaneously, the cell proliferation rate tends to decrease and patterns of cell division emerge that eventually define the histology of the leaves and stem (Donnelly et al., 1999). In addition to this change in size and division pattern, cells leaving the SAM domain gain a specific biochemistry. Cells in the SAM are heterotrophic and depend on imported carbon for the catabolic processes that occur in this part of the plant. Most cells adjacent to the SAM rapidly gain autotrophic capability by differentiation of small proplastids into chloroplasts (Fleming et al., 1996). This involves the synthesis and assembly of various components of the photosynthetic apparatus. Despite our in-depth knowledge of photosynthesis, the process of proplastid differentiation remains essentially a black box. Most work in this area has focused on the transdifferentiation of etioplasts into chloroplasts and has revealed key insights into the role of light in regulating the assembly of functional chloroplasts (for review, see Strand, 2004). It is, however, very unclear to what extent environmentally triggered etioplast differentiation relates to developmentally controlled proplastid differentiation. Exposure of the SAM to light does not trigger chloroplast differentiation in these cells (Fleming et al., 1996), indicating a fundamental difference in the situation with etioplasts.
With respect to differentiation in general, one key aspect that has emerged over the last few years is the potential role of the retinoblastoma protein (pRb) as part of the switching process by which cells cease proliferation and enter a phase of differentiation (Inzé, 2005
With respect to plants, sequencing data and cloning experiments indicate that plants possess genes that encode retinoblastoma-related (RBR) proteins (Xie et al., 1996 In this article, we show that local and transient overexpression of RBR protein in the SAM is sufficient to trigger cells toward a more differentiated state, including elements of proplastid differentiation. These data are consistent with the emerging paradigm of RBR protein as an important nexus in plant meristems for the decision to either maintain a meristem state or to enter a pathway of differentiation.
Generation of Transgenic Plants in Which RBR Gene Expression Can Be Transiently Induced
A series of transgenic tobacco (Nicotiana tabacum cv Samsun) plants was generated in which the expression of a full-length cDNA encoding RBR protein from Arabidopsis (Ebel et al., 2004
Reverse transcription (RT)-PCR analysis of leaf discs taken from a number of independent transgenic tobacco lines with or without AhTet induction led to the identification of two lines in which AtRBR transcripts were not detectable in uninduced tissue but reached high levels following induction (Fig. 1A
). These two lines (Tet::RBR3a and Tet::RBR4a) were taken for further characterization by western-blot analysis. Following induction, a low, but detectable, level of RBR protein was detectable within 4 h of treatment with AhTet (Fig. 1B). No signal was detectable in noninduced tissue samples or in induced samples of control plants engineered to contain the
To further investigate the temporal change in RBR gene expression in transgenic tobacco plants, a series of microinduction experiments were performed in which the entire SAM of Tet::RBR plants was induced with AhTet, and the level of both the endogenous NtRBR mRNA and the induced AtRBR mRNA was quantified using real-time PCR. The results (Fig. 1C) indicate that, following this manipulation, there was massive induction of AtRBR gene expression relative to an actin transcript used as an internal control. The timing of the peak of induction varied between 2 and 8 h after apex manipulation, depending on the individual apex analyzed. In all cases, the relative level of AtRBR mRNA returned to an approximately preinduction level within 16 h of the manipulation. Analysis of the mRNA level for an endogenous NtRBR gene indicated that, in the hours following induction, there was a general trend for the expression of this gene relative to the actin control transcript to decrease (Fig. 1C), but, as observed with AtRBR, within 16 h the relative level of the NtRBR transcript had returned to that observed prior to induction. This relative decrease in the NtRBR mRNA level may indicate a mechanism whereby a transient high level of the AtRBR gene product feeds back to the endogenous RBR gene to decrease its expression. Alternatively, due to the similarity of the Arabidopsis and tobacco RBR gene sequences, some limited RNA interference effect cannot be excluded. These observations on the transient nature of the induction of AtRBR gene expression were supported by in situ hybridization analysis (Fig. 1D). In control apices, there was uniform expression of the endogenous NtRBR transcript throughout the apical meristem and in the young leaf primordia. Hybridization of sections from such uninduced apices revealed no signal with a probe specific for the AtRBR transcript. Within 2 h of induction of Tet::RBR apices, a distinct signal was apparent in sections hybridized with the AtRBR probe (Fig. 1D). However, by 24 h, only a background signal was apparent in such sections hybridized with the AtRBR probe. In contrast, hybridization with the NtRBR probe of equivalent sections taken at similar time points from induced Tet::RBR apices revealed a relatively constant and uniform pattern of transcription (Fig. 1D). No overt decrease in the NtRBR mRNA level was observed during this time course (in contrast to the RT-PCR data shown in Fig. 1C), but this might simply reflect the poor quantitative nature of the in situ hybridization technique. Taken together, the western-blot data showing an increased accumulation of RBR protein and the in situ hybridization and quantitative RT-PCR data showing a massive and transient increase in AtRBR mRNA level indicate that the microinduction process led to a transient increase in RBR protein activity in the apex target tissue.
To investigate the outcome of local, transient induction of AtRBR gene expression, aliquots of lanolin impregnated with AhTet were positioned onto either the I1 or I2 position of apical meristems of Tet::RBR plants and the apices regenerated. The I1 position marks the group of cells that will normally form the next leaf primordium, whereas cells within the I2 position will not normally become incorporated into a leaf until those at I1 have done so. Growth of the apices was observed at various time points up to 5 weeks by counting leaves and imaging plants. Control experiments included both the use of mock-induced Tet::RBR apices and the AhTet induction of apices dissected from Tet::GUS plants containing the GUS reporter gene under AhTet-inducible transcriptional regulation (Pien et al., 2001 As shown in Figure 2 , local induction of the meristems of Tet::RBR apices led to dramatic retardation of plant growth. A range of phenotype was observed that allowed plants to be scored as showing essentially total repression of growth (no new leaves formed; Fig. 2, A and B), moderate inhibition of growth (four to five leaves formed; Fig. 2, C and D), or essentially no inhibition of growth relative to that observed in AhTet-induced Tet::GUS apices or mock-induced Tet::RBR plants (seven to nine leaves formed; Fig. 2, E and F). Independent of whether induction was performed at the I1 or I2 position of the meristem, 12/46 (26%) of induced Tet::RBR apices showed severe growth inhibition (Table I ). Such severe growth inhibition was never observed in either mock-induced Tet::RBR apices or AhTet-induced Tet::GUS apices. Moderate growth inhibition was observed in 4/28 (14%) AhTet-induced Tet::GUS apices, whereas 33/46 (72%) of Tet::RBR apices showed this phenotype after treatment with AhTet. The vast majority of mock-induced Tet::RBR apices (100%) and AhTet-induced Tet::GUS apices (86%) showed normal growth after manipulation, whereas only 2% (1/48) of AhTet-induced Tet::RBR apices displayed a normal rate of growth. These data indicate that local transient overexpression of the AtRBR protein in the tobacco SAM via microinduction was sufficient to induce an extended (5 week) repression of growth. These plants did not display any obvious symptoms of senescence. Instead, they remained green and apparently viable, but with an extremely limited rate of growth.
Microinduction of RBR Gene Expression Leads to Dramatic Changes in Meristem Cytology To investigate the nature of growth repression induced by the induction of AtRBR gene expression, we performed a histological and cytological analysis of induced and control apices. As shown in Figure 3A , the SAM of a control plant consists of outer cell layers (the tunica) surrounding an inner corpus in which cell division orientation is not uniform. The cells in the tunica are characterized by being relatively uniform in size, densely cytoplasmic, with a large central nucleus (Fig. 3B). Cells deeper in the corpus tend to be larger and show the first signs of vacuolation. After microinduction of RBR gene expression in the meristem, the first visible change in cytology occurred within 24 h (Fig. 3, C and D). In the area of induction, cells in the outer tunica layers became enlarged and a chain of vesicles became apparent around the central nucleus. By 72 h, the meristem became enlarged and flattened (compare Fig. 3, E and A). There was a clear gradient of cells from the flank of the meristem to the center, with cells on the flanks being vacuolated and cells in the center of the meristem being compact and intensely stained. Closer observation of these cells (Fig. 3F) revealed that they were enlarged and showed a staining pattern distinct from that observed in the tunica of control apices (compare Fig. 3, F and B). Three weeks after induction of RBR gene expression, the meristems contained a mixture of highly vacuolated cells intermingled with smaller, more compact cells (Fig. 3, G and H). The tightly ordered pattern of cell division observed in control meristems tended to disappear and the entire appearance of the SAM was very different from that of a control SAM (compare Fig. 3, G and H, and A and B). The altered meristem histology and cytology correlated with the growth phenotype of the plants (i.e. apices showing the most severe growth retardation showed the most severe cytological and histological defects).
To assay the changes in meristem cell size, the cross-sectional areas of cells in micrographs of mock-treated and induced apices were measured (Table II ). Cells in control meristems had a mean area of 145 µm2, whereas 24 and 72 h after microinduction the cells in the tunica layers had mean areas of 209 and 205 µm2, respectively. Statistical analysis of these data indicated a significant increase in mean cell size after induction of RBR gene expression (t test; P < 0.01).
Following microinduction of RBR gene expression, changes in cytology were observed not only in the SAM, but also in the leaf primordia immediately adjacent to the induction site on the meristem. Thus, as shown in Figure 4A , 72 h following induction of RBR gene expression, the entire adaxial face of the primordium closest to the site of microinduction consisted of cells that were relatively small and cytoplasmically dense compared with cells on the abaxial face, which underwent the normal process of cell expansion and vacuolation characteristic of cells in this position at this stage of leaf development. The histology of the adaxial face of primordia in induced apices is more clearly shown in Figure 4B, which can be compared with that of tissue in this region from noninduced apices (Fig. 4C). The adaxial cells in the primordia of induced apices are smaller, lack large vacuoles, and possess a cytology more reminiscent of meristematic cells, whereas the adaxial cells of control primordia undergo cell expansion and vacuolation, with cell division orientation being maintained to generate the layered structure characteristic of the maturing leaf. Measurement of cell cross-sectional areas in the adaxial tissue of primordia from induced and noninduced apices confirmed the visual impression of altered cell size, with induced adaxial cells being significantly smaller (t test; P < 0.01) than noninduced cells (Table II).
To further analyze the cytological response to microinduction of RBR gene expression, we performed a transmission electron microscopy (TEM) analysis of the tissue. A typical cell in the LI layer of the tunica is relatively small (diameter approximately 10 µm) and contains a large nucleus surrounded by dense cytoplasm within which a few small vacuoles and proplastids are visible (Fig. 5A ). After induction of RBR gene expression, significant changes in cytology were apparent within 24 h (Fig. 5B). The most noticeable change was an agglomeration of vacuoles to form larger bodies and an increase in cell size. In addition, electron-opaque, globular bodies were frequently observed within the vacuoles (which may represent lipid bodies) and electron-dense bodies were apparent within the cytoplasm. Already at these early stages of response to RBR gene induction, changes in plastid differentiation were detectable and these became obvious within 72 h (Fig. 5C). Lamellae were observed to form within the proplastids, which also tended to accumulate large electron-opaque starch granules, as well as electron-dense bodies. Such plastids were never observed in cells in control apices at this position within the meristem. The structure of the differentiating plastids within the induced meristems can be compared with plastids in a normally developing leaf primordium (Fig. 5D). At this stage of leaf differentiation, the plastids contain stacks of lamellae and prominent starch granules. Electron-dense bodies are not observed in these differentiated plastids. The cells themselves also contain relatively large vacuoles.
As shown in Figure 4, one result of induction of RBR gene expression was the accumulation of small cytoplasmically dense cells on the adaxial face of developing leaf primordia adjacent to the site of microinduction. TEM analysis confirmed the data obtained by light microscopy, as shown in Figure 5F. The adaxial cells of induced Tet::RBR apices were smaller, less vacuolated, and more cytoplasmically dense than comparable cells in this position from noninduced tissue (Fig. 5E).
A paradigm of RBR function is that it controls entry into the cell cycle via regulation of passage of cells through a point in the G1 to S phase transition. To investigate the outcome of local induction of RBR gene expression on cell division, we performed a series of in situ hybridizations using marker genes for various phases of the cell cycle (Fig. 6
). In nontreated control apices, histone H4 mRNA accumulates in spots throughout the SAM and young leaf primordia, marking cells in the S phase (Fig. 6A). Within 24 h of RBR gene induction, transcripts for H4 were virtually undetectable and did not accumulate in their characteristic speckled pattern (Fig. 6B). After 72 h, a slight recovery in H4 mRNA signal was visible in some apices, with a few cells showing a low level of transcript accumulation (Fig. 6C). Nicta;CYCA3;2 (encoding an A-type cyclin that can also be used as a marker for the G1 to S phase transition; Wyrzykowska et al., 2002
Nt;CYCB1 transcripts (encoding a B-type cyclin) accumulate in plant cells during the G2 to M phase transition. In untreated apices, Nt;CYCB1 transcripts accumulated in a speckled pattern throughout the SAM (Fig. 6D). Within 24 h of RBR gene induction, cyclinB transcripts were virtually undetectable within the meristem (Fig. 6E). After 72 h, Nt;CYCB1 transcripts (as observed for H4 and Nicta;CYCA3;2) showed limited recovery of the normal expression pattern, but with a spectrum of signal intensity depending on the apex analyzed (Fig. 6F).
In addition to the specific expression patterns of genes encoding cell cycle-associated proteins, the SAM is characterized by a number of distinct patterns of gene expression that can be used as diagnostics of meristem function. For example, transcripts encoding homeodomain transcription factors of the KNOTTED class (KNOX genes) accumulate throughout the SAM but are excluded from the region involved in specification of a new leaf primordium (for review, see Tsiantis and Hay, 2003
Meristem cells are nonphotosynthetic and do not normally express genes encoding key components of this metabolic pathway, such as the small subunit of Rubisco (RBCS; Fleming et al., 1996
As a final control to investigate the general pattern of transcriptional activity in the responding meristems, we performed hybridizations with a probe against mRNA encoding a translation initiation factor, NteIF4A. The encoded protein is involved in protein translation and can be used as a probe for general transcriptional/translational activity (Mandel et al., 1995
RBR Protein and Control of Meristem Cell Differentiation
Recent work on the RBR gene in Arabidopsis has implicated it both in maintenance of stemness of classically defined initial cells in the RAM and in control of differentiation of these cells (Wildwater et al., 2005
Previous investigators have indicated that RBR protein in tobacco can functionally interact with cyclinD/CDK complexes (Huntley et al., 1998
Although the experiments reported here depended on altered transcription of an introduced RBR gene, the endogenous function of RBR protein is likely to be highly dependent on posttranslational modification of the protein (Kaye et al., 1990
The results of our experiments also suggest that modification of cell activity in one part of the SAM (via RBR protein induction) leads to modification of cell activity in other parts of the SAM and, indeed, on the flanks of the leaf primordia adjacent to the induction site on the SAM. Thus, whereas cells induced to express RBR protein underwent initial steps in differentiation in the SAM, adjacent cells in the leaf primordium maintained meristematic cytology. This is consistent with the idea of a signaling system by which appropriate differentiation of cells in the leaf primordium requires a signal from a functioning SAM. In the absence of such a signal, the cells in the leaf do not undergo appropriate growth and differentiation. The mechanisms underlying these observations are at present unknown; however, a number of publications indicate that dynamic signaling events occur both within the SAM and between the SAM and the surrounding leaf primordia to coordinate growth activity (e.g. Waites and Hudson, 1995
Down-regulation of the KNOX transcript level in cells undergoing determination to form leaves, and concomitant accumulation of transcripts encoding ARP transcription factors, is a general facet of SAM function (Tsiantis and Hay, 2003
In conclusion, our data indicate that regulation of RBR protein in the SAM might play a similar role to that recently established in the RAM with respect to the control of stem cell differentiation. The function of retinoblastoma-like proteins in controlling entry into the cell cycle has long been established, but the potential role of these proteins in directing differentiation has been less well explored (Dimova et al., 2003
Plant Material and Transformation
R7 tobacco (Nicotiana tabacum cv Samsun) seedlings (a gift from A. Jones, University of North Carolina, Chapel Hill) were transformed (Pien et al., 2001
The Arabidopsis (Arabidopsis thaliana) RBR cDNA was cloned into the pBINHyg-TX vector (Gatz et al., 1992
Microinductions were performed as described (Pien et al., 2001
For RT-PCR, total RNA was extracted from 4-week-old seedlings using RNeasy columns (Qiagen). In situ hybridization was done as described (Pien et al., 2001
Statistical analyses were performed following the formula of Pfaffl et al. (2002)
Tobacco pieces were fixed in 2% (w/v) glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2, for 2 h at room temperature and postfixed with 1% (w/v) OsO4 in sodium cacodylate buffer at 4°C overnight. After dehydration with acetone at room temperature, the material was embedded in Spurr's standard epoxy resin with dibutyl phthalate and the resin was polymerized at 70°C for 19 h. Thin sections were stained with 2% (w/v) uranyl acetate in 50% (v/v) acetone for 30 min and alkaline lead citrate for 30 min (Reynolds, 1963
We thank Chantal Ebel and Luisa Mariconti (Swiss Federal Institute of Technology [ETH]) for fruitful discussions, Jean-Pierre Metraux for access to the Fribourg TEM facility, and Nikolaus Amrhein (ETH) for providing lab space and encouragement. Received May 8, 2006; returned for revision June 22, 2006; accepted June 25, 2006.
1 This work was supported by the Swiss Federal Institute of Technology-Zürich (grant to A.J.F. and W.G.) and the Swiss National Science Foundation (START Fellow to A.J.F.). 
2 Present address: Department of Plant Biology, University of Zürich, Zollikerstrasse 107, CH–8008 Zurich, Switzerland. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andrew J. Fleming (a.fleming{at}sheffield.ac.uk). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.083022. * Corresponding author; e-mail a.fleming{at}sheffield.ac.uk; fax 44–114–2220002.
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ABSTRACT
RESULTS
-glucuronidase (GUS) protein under Tet-inducible transcriptional regulation. An intense signal was observed in extracts from Arabidopsis plants engineered to overexpress a cyclinD gene that previous research had shown leads to the accumulation of high levels of the endogenous RBR protein (Dewitte et al., 2003
