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First published online October 24, 2008; 10.1105/tpc.108.059048 The Plant Cell 20:2746-2762 (2008) © 2008 American Society of Plant Biologists Conditional Repression of AUXIN BINDING PROTEIN1 Reveals That It Coordinates Cell Division and Cell Expansion during Postembryonic Shoot Development in Arabidopsis and Tobacco[W]
a Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Unité Propre de Recherche 2355, Université Paris-Sud XI, 91198 Gif sur Yvette Cedex, France 4 Address correspondence to catherine.rechenmann{at}isv.cnrs-gif.fr or a.fleming{at}sheffield.ac.uk.
AUXIN BINDING PROTEIN1 (ABP1) has long been characterized as a potentially important mediator of auxin action in plants. Analysis of the functional requirement for ABP1 during development was hampered because of embryo lethality of the null mutant in Arabidopsis thaliana. Here, we used conditional repression of ABP1 to investigate its function during vegetative shoot development. Using an inducible cellular immunization approach and an inducible antisense construct, we showed that decreased ABP1 activity leads to a severe retardation of leaf growth involving an alteration in cell division frequency, an altered pattern of endocycle induction, a decrease in cell expansion, and a change in expression of early auxin responsive genes. In addition, local repression of ABP1 activity in the shoot apical meristem revealed an additional role for ABP1 in cell plate formation and cell shape. Moreover, cells at the site of presumptive leaf initiation were more sensitive to ABP1 repression than other regions of the meristem. This spatial context-dependent response of the meristem to ABP1 inactivation and the other data presented here are consistent with a model in which ABP1 acts as a coordinator of cell division and expansion, with local auxin levels influencing ABP1 effectiveness.
Recent research has led to significant advances in our understanding of the molecular mechanism by which the growth regulator auxin is perceived by the plant and the signal transduced into a molecular output (Badescu and Napier, 2006  ; Quint and Gray, 2006). In particular, this work has led to the identification of the TRANSPORT INHIBITOR RESPONSE PROTEIN1 (TIR1) F-box factor as an auxin receptor whose binding with auxin leads to an SCF ubiquitin-ligase catalyzed degradation of auxin/indole-3-acetic acid (Aux/IAA) transcriptional repressors (Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). Mutational analysis has revealed that loss of functional TIR1 and related AFB genes leads in a combinatorial fashion to progressively more severe developmental and physiological affects, confirming that auxin signal transduction via the TIR1 receptor family represents a major pathway for auxin function (Dharmasiri et al., 2005b). However, even quadruple mutants in which all TIR1/AFB genes are mutated can form functional (although abnormal) plants. Although it is difficult to dismiss the possibility that in these mutants there is some residual TIR1/AFB function, the data suggest that auxin may also act via the function of an additional receptor (Badescu and Napier, 2006); indeed, classical data on auxin receptor biology support this idea. Recent results derived from the analysis of other plant growth factors, such as abscisic acid, also indicate that plants use a surprising variety of molecular mechanisms to perceive and transduce growth factor signals (Verslues and Zhu, 2007). Some of these may resemble standard signal transduction motifs identified in other organisms, whereas others seem to be unique to plants.
With respect to auxin, one potential receptor in addition to TIR1 is AUXIN BINDING PROTEIN1 (ABP1). A substantial body of evidence demonstrates that this protein binds auxin at physiologically relevant levels, that it can discriminate between physiologically active and inactive forms of auxin, and that its activity mediates the activation of ion fluxes across the plasma membrane in response to auxin (Venis et al., 1992
One approach to this problem is to generate plants in which the activity of ABP1 can be conditionally repressed. In previous work, ABP1 activity was blocked in BY2 suspension cultured cells by overexpression of a recombinant antibody fragment (scFv) directed against ABP1 (David et al., 2007 In this article, we report on the outcome of conditional repression of ABP1 activity during shoot postembryonic development using both antisense and cellular immunization approaches, the latter being performed in both Arabidopsis and tobacco (Nicotiana tabacum). These experiments indicate that ABP1 is required for the maintenance of shoot and leaf growth. In addition, by restricting the spatial and temporal parameters of ABP1 inactivation (via use of a microinduction technique) we show that ABP1 is required for the coordination of cell division and cell expansion in a highly context-dependent manner. In particular, during the very early stages of leaf formation even transient loss of ABP1-mediated coordination of cell plate formation and cell expansion has drastic downstream consequences for growth of the whole plant. These data demonstrate the requirement for ABP1 during shoot postembryonic development and accentuate the importance of developmental context in understanding the function of a gene product involved in growth factor perception.
Ethanol-Inducible Inactivation of ABP1 Leads to Repression of Plant Growth Our previous work described the generation of a number of transgenic lines of tobacco BY2 cells containing constructs that encoded single chain fragment variable regions (scFv12) derived from a hybridoma line expressing the well-characterized anti-ABP1 monoclonal antibody mAb12 (Leblanc et al., 1999b ; David et al., 2007). Analysis of these transgenic cell lines showed that the induced scFv12 protein interacted in vivo with ABP1 and led to a block on cell division.
To establish a system to conditionally downregulate ABP1 activity in the intact plant, we generated a number of transgenic Arabidopsis lines containing scFv12 constructs in a vector designed to allow induction of the encoded protein by exposure of the plants to ethanol vapor (Roslan et al., 2001
As shown in Figures 1A and 1B
, exposure of p35S:AlcR>pAlcA:SS12K (further referred to as AtSS12K) and p35S:AlcR>pAlcA:ABP1AS (further referred to as AtABP1AS) plants to ethanol led to a rapid accumulation of the scFv12 mRNA and ABP1 antisense RNAs, respectively, with transcripts being detectable within 2 h and rising to a maximum level after 24 h. Protein gel blot analysis revealed a detectable accumulation of the scFv12 protein in the AtSS12K plants after 8 h (data not shown) and strong accumulation after 24 h (Figure 1C). In antisense plants, 3 to 4 d of exposure to ethanol led to loss of detection of ABP1, even using n-butyl alcohol solubilized membrane fractions that are enriched in ABP1 (Figure 1D). Pull-down experiments using an anti-ABP1 antibody revealed that the scFv12 protein generated within the plant tissue was associated with endogenous ABP1 protein (C. Perrot-Rechenmann, unpublished data). These observations are consistent with our previous work in which the scFv12 construct was expressed in tobacco BY2 cells and where specific interaction of the scFv12 protein with endogenous ABP1 was also demonstrated (David et al., 2007
Subsequent to ethanol exposure, both scFv12 expressing lines and ABP1 antisense lines showed a dramatic reduction in growth and development of young seedlings compared with induced control seedlings (Figures 1E to 1G). Four-day-old AtSS12K seedlings exposed to ethanol exhibited small and epinastic cotyledons and the absence or delayed emergence of pale primary leaves that did not develop further (Figure 1F). Similar phenotypes were observed in seedlings in which the ABP1 antisense construct was induced by exposure of the plant to ethanol (Figure 1G). No effect of ethanol was observed in wild-type plants (Figure 1E) or in plants expressing a β-glucuronidase (GUS) reporter gene construct under the same ethanol-inducible system (see Supplemental Figure 1A online). Thus, using two different approaches to inducibly decrease endogenous ABP1 activity produced essentially the same phenotype.
Germination and growth of seedlings on soil allowed a controlled exposure to ethanol (and thus repression of ABP1 activity) at various time points. Induction with ethanol at very early stages of seedling development led to the phenotype as described in Figure 1, leading to death of the plant. When older (12-d-old) AtSS12K or AtABP1AS plants were exposed to ethanol vapor, down-curling of leaves occurred accompanied by growth arrest within a few days (Figures 2C and 2D
), whereas induced wild-type and AlcAGus control plants or noninduced AtSS12K plants showed no alteration in growth (Figures 2A and 2B; see Supplemental Figures 1B to 1D online). Extended exposure to ethanol caused drastic growth defects characterized by the formation of small, curled leaves (Figures 2C to 2G; see Supplemental Figure 1E online). Thus, when plants that had formed five visible leaves under noninducing conditions were exposed to ethanol, the 6th leaf reached a length of 9 to 11 mm over the subsequent 14 d, whereas in the same time the 6th leaf from a plant kept under noninducing conditions attained a length of 27 to 30 mm. In addition to this general reduction in growth, leaf morphology was also affected. Preexisting leaves induced to repress ABP1 activity exhibited warped and shorter laminas, whereas leaves that appeared subsequent to repression of ABP1 activity displayed a decrease both in lamina width and length. In addition, these leaves were epinastic (Figure 2H). Measurements of auxin content in shoots of ABP1 inactivated plantlets revealed that the induced phenotype was not correlated with a global modification of auxin content (see Supplemental Figure 2 online). Organization of leaf vasculature was almost normal, although rosette leaves with decreased growth exhibited a reduced number of veins (Figure 2I) (Alonso-Peral et al., 2006
Previous investigations of the effect of overexpressing ABP1 indicated that although there was little change in leaf growth rate or form, the cell size in certain regions of the leaf was altered (Jones et al., 1998 ). To investigate whether the observed leaf growth phenotype in the ABP1 inactivated plants was related to an altered internal structure of the leaf we performed a histological and scanning electron microscopy analysis. Histological analysis of leaves from ethanol-exposed AtSS12K plants indicated that all expected cell types were present and that these were arranged in an appropriate fashion compared with the wild type (Figures 3A to 3E ) (i.e., leaf adaxial/abaxial differentiation appeared normal). The severe epinasty of ethanol-induced AtSS12K leaves is clearly visible in transverse sections (Figures 3C and 3D). Some relatively minor changes in histology were apparent, for example, the occurrence of some smaller epidermal cells (Figure 3E). Occasionally, the distal tip of the induced AtSS12K leaves exhibited a lack of vascular tissue (see Supplemental Figure 4A online), indicating that the mid-rib did not extend to the tip of these leaves. In addition, scanning electron microscopy analysis revealed the occurrence of double vascular mid-ribs at the tips of a number of induced AtSS12K or AtABP1AS leaves and twisted veins (see Supplemental Figures 4B and 4C online).
Scanning electron microscopy analysis of the induced AtABP1AS leaves indicated that epidermal cell size and shape were significantly altered compared with induced control leaves of similar age (Figures 3F to 3I, Table 1 ). This analysis was somewhat constrained by the extreme curvature in the induced AtABP1AS or AtSS12K leaves, which made it difficult to view the leaf surface. Cell surface measurements were performed on the medial part of each leaf analyzed, both on the adaxial and the abaxial surfaces. Comparison of wild-type leaves with AtABP1AS leaves of similar age (leaf number 5) that were formed subsequent to ethanol exposure showed that the mean cell surface area was over threefold smaller in the adaxial epidermis of the AtABP1AS leaves and over fourfold smaller in the abaxial epidermis of the AtABP1AS leaves (Table 1). This reduction in mean cell surface area was accompanied by much slower growth of the leaves and by epinasty (decreased abaxial growth relative to the adaxial growth). With respect to the number of cells per leaf, there was approximately half the number of epidermal cells in an ethanol-induced AtABP1AS leaf compared with a wild-type leaf of equivalent size (comparison of leaf number 5 and 9 from AtABP1AS and wild-type plants, respectively; Table 1). Thus, both tissue expansion and cell division were altered in leaves of ABP1-inactivated plants.
To investigate further the effect of ABP1 inactivation on cell division, we analyzed the expression of core cell cycle regulators (Figure 4A ). ABP1 inactivation was followed by a rapid decrease in mRNA accumulation of D-type CYCLINS (Figure 4A), which are early regulators of the G1/S transition (Menges et al., 2006 ). In the same time frame, the transcriptional activator E2Fb was not significantly changed, whereas the transcriptional repressor E2Fc was transiently increased, as were transcripts of the retinoblastoma-related gene, which is thought to act at the interface between regulators of the cell cycle and the cell cycle process itself. Accumulation of the G2/M marker CYCB1.1 mRNA was not significantly affected within the first 24 h of ABP1 inactivation but tended to decrease later. As CYCB1.1 is also strictly regulated at the protein level and is often used as a quantitative marker of cell division, we introgressed the pCYCB1.1:DboxCYCB1.1-GUS reporter (Colon-Carmona et al., 1999) into the SS12K line. After 48 h of ethanol induction, GUS staining was decreased in young leaves and at the apical meristem, consistent with a decrease in cell division frequency (Figures 4B to 4E).
Normally during Arabidopsis leaf development, there is a progressive entry into endoreduplication, with developmentally older leaves gradually containing more cells with higher nuclear C values. This increased nuclear C value generally correlates with a larger cell size (Sugimoto-Shirasu and Roberts, 2003 ). We therefore investigated the outcome of reduced ABP1 activity on nuclear C value in control and AtSS12K leaves before or after exposure to the inducer (Figures 3J to 3L). In this experiment, plants were grown under noninducing conditions until leaf number 5 became visible ( 1 mm length). The plants were then exposed to ethanol vapor and the plants allowed to grow for a week until appearance of leaf number 9. The C value for nuclei extracted from leaf numbers 3, 4, and 6 were then obtained. Leaf number 3 of each genotype tested (the wild type or AtSS12K) had a similar spectrum of DNA content/nucleus after exposure of the plant to ethanol (Figure 3L), with C values ranging from 2C to 32C, with a median value of 8C. However, both leaves 4 and 6 (which developed during the exposure of the plant to ethanol) showed an altered spectrum of DNA content/nucleus after induced repression of ABP1 activity (Figures 3J and 3K). In both cases, AtSS12K leaves had a maximum of 4C content and 20% of 2C cells, whereas wild-type leaves contained more cells with a higher C content. For example, wild-type leaf 4 contained a large proportion of 16C cells, with >10% of the cells having a 32C content. These data show that after ethanol induction, the nuclei in the AtSS12K leaves maintained a much lower C value than developmentally equivalent leaves formed under noninducing conditions, suggesting that ABP1 is necessary to promote endoreplication. As described above, the cells formed in these induced leaves were smaller than cells in developmentally equivalent leaves; thus, there was a correlation between mean nuclear C value and cell size.
Local Repression of ABP1 in the Shoot Apical Meristem Reveals Its Context-Specific Role in the Coordination of Cell Division and Growth
We particularly focused on the SAM since auxin has been implicated in the earliest events of leaf initiation in this tissue (Reinhardt et al., 2003
In a first series of microinduction experiments, apices were dissected to reveal the SAM. Lanolin beads impregnated with AhTet were then positioned on either the I1 position of the SAM (where leaf initiation is imminent) or on the I2 position opposite (where leaf formation does not normally occur until after leaf formation at the I1 position) (see Figure 6A for delineation of I1 and I2 sites). AhTet induction at the I1 position led to a marked retardation of apex growth (as indicated by limited leaf initiation and growth over the 4 weeks subsequent to the induction process) (Figure 5D). By contrast, microinduction of NtSS12S6 SAMs at the I2 position did not generally lead to a retardation of apex growth, with leaves being initiated and growing out in a fashion comparable to that observed in control mock-induced plants (Figure 5E). Analysis of a total of 96 apices (with 59 being induced at the I1 and 37 being induced at the I2 position) revealed a statistically significant different frequency (0.01 confidence level) of apex growth response when induction at the I1 or I2 position was compared (Table 2 ). Severe reduction of growth was not observed when the I1 of control Tet:GUS apices (harboring the GUS reporter gene under tetracycline-dependent transcriptional control) (Pien et al., 2001 ) was induced (Table 2), indicating that the observed growth retardation was linked with the induction of the scFv12 gene rather than with the induction process itself.
To further investigate the nature of this differential response to induction of scFv12, we performed scanning electron microscopy and histological analysis of induced and noninduced apices. Scanning electron microscopy analysis indicated that subsequent to scFv12 induction at the I1 position, leaf initiation still occurred; however, the rate of primordium outgrowth was drastically retarded relative to control treated apices (Figure 6). Thus, after induction at the I1 position in NtSS12S SAMs, a primordium bulge became apparent 2 d later (Figure 6C), but the primordium remained small even at 10 d after induction (Figure 6E). New primordia were formed subsequently at the appropriate position on the SAM, but the growth rate of these primordia was extremely slow. Measurement of cell surface areas in induced and noninduced regions on the SAM indicated that there was a slight but significant (0.05 confidence level) increase in the mean cell surface area in the induced I1 area relative to the equivalent position on a noninduced SAM (Figures 6B, 6D, and 6F, Table 3).
In addition, while the I1 position is normally characterized by a regular cobblestone cell pattern (Figure 6B), within 24 h of induction of the scFv12 construct cells in this area showed a more irregular division pattern (Figure 6D).
Histological analysis of the I1-induced NtSS12S SAMs revealed a loss of the normal cellular organization at the induced position prior to bulge formation (Figures 7A and 7B ). The SAM is normally characterized by a distinct layered organization in which cell division pattern is constrained in the outer regions of the SAM so that new cell walls form perpendicular to the surface of the SAM. This results in the formation of cell layers toward the surface of the SAM (the tunica) below which the orientation of cell division plane is not so constrained (leading to the definition of the corpus). After induction of a NtSS12S SAM at the I1 position, this tunica organization was lost due to the occurrence of cytokinesis in abnormal planes (Figures 7A and 7B). In addition, there was a marked and significant decrease (t test: 0.01 confidence level) in cell area in these longitudinal sections at the I1 induced region relative to equivalent I1 regions of control SAMs (Table 4). At the same time, the cell cross-sectional area in the I2 region of the I1-induced SAMs was larger than the equivalent I2 region of control SAMs (t test: 0.01 confidence level; Table 4). Thus, as a result of the I1 induction of scFvABP1, there was both a disorganization of the cellular patterning at the I1 position and there was an increase in mean cell size at the I2 position (with cellular pattern remaining approximately normal).
As described above, despite the induction of a disorganized cellular pattern at the I1 position in the NtSS12S SAMs, a delayed leaf initiation still occurred. Although the histology of the induced organs appeared relatively normal with, for example, clear differentiation of vascular tissue, cellular organization at the distal tip of the primordia appeared abnormal (Figure 7C). In particular, although in normal primordia the elongated cells characteristic of the emerging vasculature extend to the tip of the primordium (Figure 7D), in the tissue derived from the induced portion of the NtSS12S SAMs overt vascular differentiation seemed to terminate below the tip of the primordium (Figure 7C). After initiation, the subsequent growth of the leaves derived from the induced NtSS12S SAM tissue was greatly decreased. Thus, while control leaf primordia underwent a process of extensive elongation (Figure 7F), leaves derived from NtSS12S induced SAM tissue remained relatively short (Figure 7E). To further analyze these cellular defects, we performed qRT-PCR analysis using dissected tobacco apices, both with and without suppression of ABP1 activity. We focused on a number of cell cycle marker genes known to act at various phases of the cell cycle (see Supplemental Figure 5 online). As already observed in Arabidopsis leaves (Figure 4), transcript levels for D-type cyclins rapidly decreased after inactivation of ABP1 but in these experimental conditions was followed by a partial recovery in transcript level. Histones H1 and H4 and PCNA (all S-phase markers) showed a relative rise in transcript level 24 h after repression of ABP1 activity, as did a marker for the M phase of the cell cycle (CYCLIN B). Interestingly, transcripts for a RETINOBLASTOMA RELATED PROTEIN (whose activity links D-type cyclins and the G1/S phase transition) showed a temporal transcript pattern similar to the S phase marker genes and distinct from the D-type cyclin genes. To investigate the spatial pattern underlying these changes in cell cycle gene expression, we performed a series of in situ hybridizations of tobacco apices in which ABP1 activity had been suppressed. In control apices, the ABP1 transcript is uniformly expressed (see Supplemental Figures 6A and 6B online) and the meristem shows a characteristic pattern for the NTH15 (KNOX-like) marker gene in which transcripts are absent from the I1 position (see Supplemental Figure 6C online). The general pattern of cell cycle gene expression in the SAM was little changed after repression of ABP1 activity (data not shown). However, after suppression of ABP1 activity at the I1 site, two regions of decreased NTH15 transcript level become apparent (see Supplemental Figure 6D online), suggesting that the timing of leaf initiation was altered so that two regions within the meristem underwent commitment to leaf formation.
The primordia originating from the induced I1 of NtSS12S apices were rounder than wild-type primordia, and this was associated with an altered pattern of vasculature (see Supplemental Figure 7 online). Thus, instead of the clearly defined single mid-rib observed in control tobacco leaves, two or more parallel veins formed toward the center of the induced leaves (see Supplemental Figure 7A online). This alteration in vascular pattern and leaf shape was reminiscent of that reported in other plants to result from supply of the auxin transport inhibitor naphthylphthalamic acid (NPA) (Mattsson et al., 1999 To investigate whether induction of NtSS12S leaves at later stages of development led to changes in leaf form, microinductions were performed along the flanks of otherwise normally formed leaf primordia. These inductions led to a range of growth response varying from a waviness along the leaf lamina to downward curling of the leaf lamina, reminiscent of the phenotype observed in Arabidopsis (see Supplemental Figure 7C online). In all these cases there was no overt abnormality in the pattern of leaf vasculature.
Repression of ABP1 Leads to Rapid Changes in Auxin Signaling-Related Transcript Levels
These data are important since they indicate that ABP1 is required to maintain appropriate expression of Aux/IAA genes in leaf tissue. In addition to the auxin/TIR1-mediated derepression of these genes, ABP1 also plays a role (Mockaitis and Estelle, 2008 ).
ABP1 has long been identified as a potentially important mediator of auxin action. Understanding its role in the plant requires characterization of the loss-of-function phenotype during development. However, constitutive loss of ABP1 activity is embryo-lethal, thus precluding any firm conclusions on the requirement of ABP1 during postembryonic development (Chen et al., 2001 ). In this article, we provide data characterizing the role of ABP1 during vegetative plant growth. These results were obtained using both a novel cellular immunization technique to conditionally repress ABP1 activity and an antisense approach to conditionally decrease ABP1 protein level. Both approaches led to broadly similar phenotypes, suggesting that in both cases the phenotypes observed were indeed due to a loss of ABP1 function. In addition, by combining techniques to conditionally suppress ABP1 activity with a microinduction method to allow spatial/temporal control of gene expression, we show that the requirement for ABP1 activity in the intact plant is highly context dependent and that the outcome of loss of ABP1 activity in shoots involves aspects of both cell division and expansion. As well as providing key data on the endogenous function of ABP1, our results demonstrate the importance of temporal and spatial context in understanding the role of mediators of plant development.
ABP1 Is Required during Postembryonic Development and Acts at the Interface of Cell Division and Expansion in Leaf Morphogenesis
Endoreduplication can be understood as a truncated cell cycle in which the mitotic phase is skipped, and it is believed that the regulatory machinery required for the G1/S transition and DNA replication is shared by both the mitotic cell cycle and endocycle (Vlieghe et al., 2007
ABP1 Is Required during Early Leaf Initiation and Acts on Cellular Patterning and Cell Growth In an early step of leaf initiation, founder cells are recruited from the peripheral zone located at the flanks of the SAM. The outer region of the SAM is characterized by an ordered pattern of cell division, forming the tunica, which surrounds an inner mass of cells, the corpus. Transient repression of ABP1 activity at the I1 site of the SAM (the position of presumptive leaf formation) led to disruption of this cellular patterning. Cell size was not massively changed; rather, there was a change in cell shape and the positioning of the new cell plates laid down as cells in this region underwent cytokinesis. Interestingly, these data are reminiscent of the altered cellular patterning reported in the ABP1 knockout mutant (Chen et al., 2001 ). Repression of ABP1 activity after leaf initiation did not lead to a significant alteration of tissue organization, suggesting that the influence of ABP1 on cell patterning might be highly context dependent. Both the embryo and the SAM are distinguished by the presence of relatively undifferentiated cells, suggesting that a role of ABP1 in coordinating cell plate formation might be limited to this cell type.
Although repression of ABP1 led to a cellular patterning defect at the I1 position of the SAM, the same manipulation at the I2 position (where leaf initiation is due to occur subsequent to that at the I1) did not have any outcome on cellular patterning or growth of the tissue, indicating that not all cells in the peripheral zone are equally responsive to altered ABP1 activity. Although we cannot discount the possibility of differential posttranscriptional activation of ABP1, recent investigations into the role of auxin in leaf initiation have led to the paradigm that local, transient maxima of auxin level occur at the site of leaf initiation (I1), whereas relative depletion of auxin occurs at the distant I2 position (de Reuille et al., 2006
Although unambiguous support for this paradigm will require direct quantitation of auxin levels within the SAM (a technically challenging requirement), our data do provide evidence that ABP1 mediated response maxima correlate with the auxin maxima, itself tied with leaf initiation. The observation that repression of ABP1 activity at the I1 site led to an altered cellular patterning is also consistent with the idea that an influence of ABP1 on cell division might only be apparent in regions of relatively high auxin level. In the ABP1 knockout mutant, the phenotype was also characterized by a loss of appropriate cellular patterning during early stages of embryo development when relatively high levels of auxin are predicted to occur (Chen et al., 2001 Interestingly, the altered cellular patterning at the I1 position induced by repression of ABP1 was associated with a prolonged suppression of expression of the KNOTTED-1-like transcription factor NTH15. The exact relationship between auxin and the key developmental regulators of meristem function, such as the KNOTTED class of homeodomain transcription factors, has been the subject of a number of investigations. The data reported here add to this body of literature, but the mechanism of interaction remains obscure. Our results indicating a link between ABP1 and expression of early auxin-responsive genes of the Aux/IAA family provide one potential way by which ABP1 and transcriptional control of developmental regulators might interact. Although ABP1 clearly has the potential to influence plant function via non-nuclear targets, the data presented here indicate the interaction of ABP1 activity and auxin-associated gene expression. The mechanism by which ABP1 affects expression of Aux/IAA genes needs to be elucidated. The absence of effect of ABP1 inactivation on the level of free auxin within the same time frame (see Supplemental Figure 2 online) indicates that the decrease in Aux/IAA transcripts does not result from a change in auxin content but rather from an altered regulation of theses genes at the transcriptional or posttranscriptional level. The data presented here show that decreased ABP1 activity leads to an array of altered transcript levels for genes associated with the TIR1 auxin signaling pathway. The contribution of ABP1 to the fine-tuning of early auxin-responsive gene regulation suggests a crosstalk with the SCFTIR1 pathway, and elucidating how ABP1 interacts with this pathway will be an important challenge of the coming years.
Altered Cellular Patterning via Repression of ABP1 Activity Leads to Downstream Events That Alter Plant Growth and Form
Published data clearly implicate auxin flux as having a major role in vascular patterning (Scarpella et al., 2006
The ability of plant tissue to respond to (and cope with) altered cellular patterning is also demonstrated by the observation that the changes in cellular patterning at the I1 position of the SAM induced by local repression of ABP1 activity was to some extent compensated for by altered cell growth at the I2 position (i.e., mean cell size at the I2 position increased after the inactivation of ABP1 at the I1 position). The mechanism of this local growth compensation is unclear, but the paradigm of autoregulated auxin fluxes provides an attractive mechanism by which a local change in cell growth and patterning involving altered auxin signaling might automatically lead to altered signal flux in a region at a distance from the initial perturbation to elicit a compensatory growth response. The application of recent modeling approaches to understanding dynamic changes in auxin flux and their influence on leaf primordia formation (de Reuille et al., 2006 In conclusion, the data reported here show that ABP1 activity is required during postembryonic development. In particular, they suggest that the role of ABP1 is highly context dependent and that, depending on auxin level and differentiation state, ABP1 can act on the cell cycle, endocycle, cell plate formation, and cell expansion and contributes to the control of auxin-related gene expression. The mechanism by which ABP1 acts in the coordination of these critical cellular parameters is a matter for urgent consideration.
Plant Material and Growth Conditions The Columbia-0 ecotype of Arabidopsis thaliana was used for transformation and experimental controls. Line R7 of Nicotiana tabacum, cv Wisconsin, overexpressing the Tet repressor, was used for transformation and for controls. Seeds were surface sterilized, stratified for 3 d at 4°C, and germinated on plates containing half-strength Murashige and Skoog (MS) basal salt mixture, buffered at pH 5.8 with 2.5 mM 2-(N-morpholino) ethanesulfonic acid and 0.9% vitro agar (Kalys). Square Petri dishes were used and placed in a vertical position at 22°C under constant light at 99 µmol·m–2·s–1 intensity. Culture in soil was performed in greenhouses for plant selection and seed production and in environmental growth chambers at 22°C constant, 16 h daylight, and 65% hygrometry for inductions and phenotype studies.
Induction of scFv and ABP1 Antisense Expression
In tobacco, for Ahtet inductions at the tissue level prior to RNA extraction and protein gel blot analysis, leaf strips were incubated overnight in half-strength MS medium, pH 5.6, containing 10 µM anhydrotetracycline. Microinductions with AhTet were performed as described (Pien et al., 2001
DNA Constructs and Transformation
Arabidopsis transformation was performed by floral dip (Clough and Bent, 1998
Tobacco transformation was performed by agroinfiltration of leaf disks as described (Horst et al., 1985
Microscopy
For scanning electron microscopy of Arabidopsis, leaves were treated with 70% ethanol and were analyzed with a Hitachi S-3000N in ESED mode. Samples were slowly frozen at –12°C under partial vacuum on the Peltier stage before observation. Cell surface measurement was made using Image J 1.034s software (NIH). For tobacco, cryo-scanning electron microscopy was as previously described (Fleming et al., 1999
qRT-PCR
Statistical analyses were performed following the formula of Pfaffl (2001)
Protein Analysis
Flow Cytometry
Accession Number
Supplemental Data
We thank K. Ljung for auxin content measurements. We acknowledge A. Jones for the R7 line; M. Lopez-Vernaza, A. Eschstruth, and F. Ouedrago for technical assistance; O. Catrice for help in flow cytometry; and S. Domenichini for scanning electron microscopy. N.B. was funded by the Ministère de la Recherche et de la Technologie; J.W. and D.C. received ACCY postdoctoral fellowships, and both labs were supported by the ACCY European Research Training Network HPRN-CT-2002-00334. During part of this work, C.P.-R. was supported by the Centre National de la Recherche Scientifique and a French ACI programme "Biologie du développement et physiologie integrative," and A.J.F. was supported by a START Fellowship from the Swiss National Science Foundation in lab space generously provided by N. Amrhein.
1 These authors contributed equally to this work. 
2 Current address: Station de Génétique et Amélioration des Plantes, Institut National de la Recherche Agronomique, Route de Saint-Cyr, 78026 Versailles Cedex, France.
3 Current address: University of Auckland, School of Biological Sciences, Auckland 92019, New Zealand. The authors 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.plantcell.org) are: Catherine Perrot-Rechenmann (catherine.rechenmann{at}isv.cnrs-gif.fr) and Andrew J. Fleming (a.fleming{at}sheffield.ac.uk).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.108.059048 Received March 4, 2008; Revision received September 22, 2008. accepted October 1, 2008.
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ABSTRACT
INTRODUCTION
light-chain signal peptide coding sequence at the 5'-end (SS prefix) and with or without a sequence encoding a KDEL extension at the 3'-end for secretion (S suffix) or retention in the ER (K suffix) (David et al., 2007
