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First published online May 5, 2006; 10.1105/tpc.106.041533 The Plant Cell 18:1383-1395 (2006) © 2006 American Society of Plant Biologists GLABROUS INFLORESCENCE STEMS Modulates the Regulation by Gibberellins of Epidermal Differentiation and Shoot Maturation in Arabidopsis[W]
a Centre for Novel Agricultural Projects, Department of Biology, University of York, York YO10 5YW, United Kingdom 1 To whom correspondence should be addressed. E-mail pb22{at}york.ac.uk; fax 44-1904-328762.
As a plant shoot matures, it transitions through a series of growth phases in which successive aerial organs undergo distinct developmental changes. This process of phase change is known to be influenced by gibberellins (GAs). We report the identification of a putative transcription factor, GLABROUS INFLORESCENCE STEMS (GIS), which regulates aspects of shoot maturation in Arabidopsis thaliana. GIS loss-of-function mutations affect the epidermal differentiation of inflorescence organs, causing a premature decrease in trichome production on successive leaves, stem internodes, and branches. Overexpression has the opposite effect on trichome initiation and causes other heterochronic phenotypes, affecting flowering and juvenile–adult leaf transition and inducing the formation of rosette leaves on inflorescence stems. Genetic and gene expression analyses suggest that GIS acts in a GA-responsive pathway upstream of the trichome initiation regulator GLABROUS1 (GL1) and downstream of the GA signaling repressor SPINDLY (SPY). GIS mediates the induction of GL1 expression by GA in inflorescence organs and is antagonized in its action by the DELLA repressor GAI. The implication of GIS in the broader regulation of phase change is further suggested by the delay in flowering caused by GIS loss of function in the spy background. The discovery of GIS reveals a novel mechanism in the control of shoot maturation, through which GAs regulate cellular differentiation in plants.
Organ initiation in plants, in contrast with animals, takes place through much of the life cycle. Successive organs may, however, develop distinctive morphological and physiological characteristics that are dependent on the developmental stage of the plant at the time the organ primordia are fated. This maturation process occurs both before and after the plant develops reproductive competence. During embryogenesis, the shoot apex produces embryonic leaves or cotyledons, which contrast with postembryonic leaves in their morphology and role in storage reserve accumulation. After germination, successive leaves go through a juvenile phase before adopting an adult form, a process termed vegetative phase change, which is largely age dependent but independent of growth rate (Telfer et al., 1997  ). Shoot maturation also affects leaves that are generated as the plant is committed to flowering, and their pattern of differentiation is thought to depend on the developmental state of corresponding primordia in the shoot apex at the time of floral induction (Hempel and Feldman, 1994; Telfer et al., 1997). In Arabidopsis thaliana, the changing distribution of trichomes, which are spiky epidermis-derived structures at the surface of aerial organs, is a robust morphological marker of phase change. During vegetative development, juvenile leaves only develop trichomes on their upper (adaxial) side, whereas adult leaves produce trichomes on their adaxial and lower (abaxial) sides. After flowering, trichomes forming on the adaxial side of inflorescence leaves (cauline leaves) follow a contrasting pattern of initiation and are gradually less abundant on successive leaves (Telfer et al., 1997).
Phase change and trichome distribution are known to be influenced by gibberellins (GAs). In maize (Zea mays), GAs promote the expression of adult traits, in particular trichome production, during the vegetative phase (Evans and Poethig, 1995 We report here the identification of a putative C2H2 transcription factor, which modulates the regulation of shoot maturation by GAs, and show that it plays a central role in epidermal differentiation through its influence on GL1 activity.
Overexpression of GLABROUS INFLORESCENCE STEMS in Arabidopsis Stimulates Trichome Initiation and Causes the Heterochronic Expression of Juvenile Traits In an effort to uncover novel functions of transcription factors in plants, a genome-wide reverse genetics analysis of gain-of-function phenotypes was conducted at Mendel Biotechnology across different Arabidopsis transcription factor families (Riechmann et al., 2000 ). This experiment identified novel genes that play a role in the control of developmental phase change and trichome production. Among them, GLABROUS INFLORESCENCE STEMS (GIS; corresponding to At3g58070) was first uncovered by screening transgenic Arabidopsis plants overexpressing transcription factor genes. Multiple independent 35S:GIS overexpression lines displayed an abnormally high density of trichomes on inflorescence organs (Figures 1A and 1B
). Two representative lines were selected for detailed studies: line 6 and line 8, which exhibited high and moderate levels, respectively, of GIS overexpression (81- and 18-fold wild-type levels). In both of these lines, the distribution of trichomes on cauline leaves was significantly altered: instead of decreasing in numbers on the adaxial side of successive leaves, trichomes remained unusually abundant, which suggested that progression of the epidermal differentiation program was delayed. In lines 6 and 8, the average adaxial trichome density on the second cauline leaf was 68 and 45% higher, respectively, than in control plants (Figure 2F
). Significantly more trichomes were also noticeable on stems and sepals in the overexpressors. In addition to suppressing the progressive decline in trichome production, GIS overexpression caused the formation of ectopic trichomes on carpels, petals, and even stamens (Figure 1B). By contrast, trichome density was normal on rosette leaves of 35S:GIS lines (Figure 2F).
35S:GIS plants also displayed a number of phenotypic changes that we interpreted as heterochronic shifts in development. For example, 35S:GIS plants flowered significantly later than wild-type plants, after producing more leaves (Table 1 ). GIS overexpression also caused the occasional appearance of aerial rosettes on the inflorescence stems of transgenic plants, in place of cauline leaves (Figure 1C).
In summary, GIS overexpressors displayed a phenotype consistent with a delay in shoot maturation, associated with a strong induction of trichome production on the inflorescence.
GIS Loss-of-Function Affects the Timing of Trichome Initiation on Inflorescence Organs
We focused our initial analysis on trichome production in the mutant. The pattern of trichome initiation is well documented for leaves but not for other aerial organs. To better assess the possible role played by GIS in epidermal differentiation, we first characterized in detail trichome distribution on stems, paraclades (secondary inflorescence shoots or branches), and flowers in wild-type plants. In addition to the known decline in adaxial trichome production on successive inflorescence leaves (Telfer et al., 1997 In contrast with the overexpression phenotype, trichome initiation was negatively affected on all inflorescence organs in gis mutants, and the effect was stronger in later than in earlier organs (Figures 2A to 2D). The reduction in trichome density was most noticeable on paraclade stems (branches), which were near-glabrous or glabrous (hence the gene name), and was also detectable on main stem internodes and cauline leaves, mostly above the first branch. The flowers of gis followed a similar trend, with a more steady decrease in trichome density. The number of branches or spikes on trichomes was also affected in the mutant. In contrast with wild-type stems, where most trichomes possessed one or two branches, an average 85% of the trichomes on gis stems had three branches and were more similar in this respect to leaf trichomes (Figure 1F). Such an increase in trichome branching was also observed on rosette leaves, most noticeably on their abaxial side (data not shown). However, in contrast with inflorescence organs, rosette leaves produced a normal number of trichomes in the mutant (see below). In addition, unlike some of the trichome initiation mutants, gis did not exhibit any obvious defect in anthocyanin, root hair, or mucilage production (data not shown). As GIS overexpression not only affected trichome production, but also induced other heterochronic phenotypes, we examined the process of shoot maturation in the mutant. We found that the mutation did not affect flowering time in long or short days, the rate of leaf initiation, or plant size (Table 1). We also examined leaf shape and venation patterns during vegetative and inflorescence development but did not find any significant difference between gis and wild-type plants. Therefore, in contrast with overexpression, GIS loss of function mainly affected epidermal differentiation after floral induction under normal conditions. To verify the GIS loss-of-function phenotype, we generated multiple transgenic lines in which the GIS gene was silenced by an RNA interference (RNAi)–based approach (see Supplemental Figure 1B online); most of these lines showed a phenotype comparable to that of the T-DNA mutant (Figure 1E). As a further verification that the phenotype was caused by the GIS loss-of-function mutation and not by another linked mutation, we overexpressed GIS under the control of the 35S promoter in the gis T-DNA line background. Constitutive overexpression rescued the mutant phenotype in most lines, also causing an increasing in trichome density on flowers and on stems (Figure 1D).
The gis Mutation Is Semidominant
GIS Acts Upstream of the Trichome Initiation Complex
To further assess the functional relationship between GIS and trichome initiation regulators, we measured the expression level of GL1, GL3, and EGL3 in developing stems and flowers of the gis mutant. As shown in Figure 4 , the expression of GL1, GL3, and EGL3 was significantly lower in the mutant than in wild-type controls. The difference was not simply the result of a decrease in the production of trichomes, where these genes are also expressed, since no significant difference in GL1 expression could be detected in ttg-1 (which is completely glabrous) and since GL3 is upregulated in this mutant (Zhang et al., 2003 ) (Figure 4). By contrast, we found that the expression of GL1 and GL3 was significantly increased in 35S:GIS plants and that their induction level correlated with the level of expression of GIS overexpression. It was not the case for TTG1 expression, which was neither affected in the overexpressing lines nor in the mutant (Figure 4). Taken together, these genetic experiments and expression studies argued that GIS acts upstream of the trichome initiation complex.
GIS Encodes a Transcription Factor of the C2H2 Family That Is Highly Expressed in Stem Epidermis and at Early Stages of Inflorescence and Flower Development Analysis of the predicted amino acid sequence of GIS indicates that it contains a C2H2 domain found in transcription factors of the TFIIIA class and is most similar to KNUCKLES and to the ZFP group of transcription factors (Figure 5 ; see Supplemental Figure 3 online) (Meissner and Michael, 1997 ; Payne et al., 2004).
To determine the pattern of GIS expression in wild-type plants, we first performed a quantitative RT-PCR analysis of transcript levels in different organs. In agreement with the phenotype of gis mutants, we found that GIS expression is low in rosette leaves and undetectable in roots. By contrast, GIS is most highly expressed in developing stems and branches, where expression levels are fairly consistent in successive paraclades (Figure 6A ). To refine our analysis, we performed in situ hybridizations using GIS-specific probes on sections of developing inflorescence stems. This experiment confirmed that the gene is expressed broadly in the inflorescence, particularly in primary and secondary inflorescence meristems. It also showed that GIS is expressed strongly in the stem epidermis and in floral meristems (Figures 6B, 6D, and 6E).
In summary, we found that the pattern of GIS expression in the inflorescence, epidermis, and trichomes is consistent with the trichome phenotypes of overexpressors and loss-of-function mutants, although it could also support a wider role in the control of inflorescence maturation that is not apparent in the mutant but is suggested by the overexpression phenotype.
Epidermal Differentiation Is Less Responsive to GAs in gis Mutants As expected, PAC applications induced late flowering and the shortening of internodes, indicating that the treatment was successful. By contrast, plants treated with GA flowered earlier and had a characteristic tall phenotype (Table 2).
We did not observe any significant difference in the mutant response to variations in GA signaling with respect to flowering time and leaf production (Table 2). However, there was a small but significant difference (t test, P < 0.001; n = 20) in the number of juvenile leaves produced by gis mutants compared with wild-type plants when high levels of PAC or GAs were applied: abaxial trichomes appeared noticeably later in the mutant on PAC- or GA-treated rosette leaves (Table 2 ), which suggested that the mutant was more sensitive to decreases and less to increases in GA levels. In line with these observations, we noticed that trichome initiation on later leaves was also less responsive to GA and more to PAC applications (Figure 7C ). PAC also inhibited trichome initiation on cauline leaves and stems more strongly in the mutant than in wild-type plants, whereas trichome production was less induced if at all by GA applications (Figures 7A, 7B, and 7D). Taken together, these observations indicated that the mutant is altered in its sensitivity to GAs and suggested a role for GIS in the epidermal expression of vegetative phase change that is only revealed when GA signaling is altered.
GIS Acts Downstream of SPINDLY and Affects the Flowering Phenotype of spindly Mutants To further probe the role of GAs in modulating its function, we investigated the interactions between GIS and components of the GA signaling pathway. We first characterized genetic interactions between GIS and SPINDLY (SPY), which encodes a putative O-linked ß-N-acetylglucosamine transferase playing a repressive role in GA signaling (Jacobsen et al., 1996 ). spy mutants display an early-flowering and accelerated growth phenotype that is largely reflective of constitutive GA response and bear many similarities with the phenotype of GA-treated plants (Jacobsen and Olszewski, 1993). They also transition between the juvenile and adult phases of vegetative development earlier than wild-type plants (Telfer et al., 1997) and produce more trichomes on rosette and inflorescence leaves (Chien and Sussex, 1996; Perazza et al., 1998). We therefore asked whether GIS was required for the expression of the vegetative and inflorescence trichome phenotypes in this mutant and constructed gis spy double mutants, using for this analysis spy-3, which is in the Col-0 background (Jacobsen and Olszewski, 1993).
We confirmed that spy-3 mutants produce leaf abaxial trichomes earlier than control plants (Telfer et al., 1997
The trichome phenotype of gis spy mutants could have been largely predicted from the observation of gis mutants to which GAs had been applied. We noticed, however, an important difference when we recorded the growth and development characteristics of the double mutant: in contrast with GA-treated gis mutants, which flowered at a similar time as GA-treated controls, gis spy mutants flowered significantly later than spy mutants (Table 1). A consistent yet smaller delay in flowering was also found in gis/+ spy heterozygotes. These observations suggested that GIS has a positive influence on flowering, independently of GA signaling, which can only be detected when repression by SPY of flowering is abolished. Interestingly, SPY is known to also act independently of GA signaling to repress flowering in long days (Tseng et al., 2004 ). To further investigate how GIS loss-of-function affects the phenotype of spy mutants, we measured GIS expression in spy inflorescence stems. As shown on Figure 8F, we found that GIS expression was significantly higher in spy-3. This was in contrast with the expression of SPY in GIS loss-of-function mutants, which we found was unchanged compared with wild-type controls (data not shown). It therefore appeared that SPY is involved, directly or indirectly, in the repression of GIS and that derepression of GIS in spy mutants is associated with an increase in trichome density. Taken together with the phenotype of gis spy mutants, these observations strongly suggested that GIS acts downstream of SPY.
GIS Is GA Inducible and Its Induction Kinetics Are Similar to Those of GL1
Since we have shown that GL1 is likely to act downstream of GIS and since GL1 expression levels are known to be regulated by GAs (Perazza et al., 1998 These experiments showed that GIS is GA inducible and that changes in its expression depend on the activity of GAI. They also indicated that the response of GL1 expression to variations in GA levels is similar in time to that of GIS and is also abolished by GAI repression.
GIS Mediates the Induction of GL1 by GAs To further examine the role played by GIS in the regulation of GL1 expression, we also compared GL1 transcript levels in developing inflorescence shoots of gis, gis spy, and spy mutants. Consistent with the effect of GA applications on GL1 expression, GL1 transcripts were significantly more abundant in spy-3 than in wild-type plants. By contrast, GL1 was more weakly expressed in gis spy mutants, and its expression level was comparable in the double mutant and in gis (Figure 8F). Taken together, these results suggested that GIS mediates the induction of GL1 expression by GAs in inflorescence shoots.
Repression of Trichome Initiation Involves GAI and Is Independent of GIS Expression Levels
Trichome initiation is known to require normal levels of GA signaling, since the ga1-3 mutant, which is severely deficient in the production of GAs, is near-glabrous. The gai signaling mutant is also deficient in trichome production, especially on inflorescence organs (Figure 3D), although it still produces leaf trichomes. In addition to its effect on initiation, GA deficiency also leads to a drop in GL1 transcript levels (Perazza et al., 1998 To this end, we first measured GIS expression in developing inflorescence shoots of the ga1-3 and gai mutants and found that, in agreement with our hypothesis, GIS is expressed at significantly lower levels in the mutants than in wild-type plants. We also found that GL1 was repressed in both mutants (Figure 8E). To test whether GIS downregulation is necessary for repression of trichome initiation, we overexpressed GIS in the ga1-3 and gai mutants. Surprisingly, we found that GIS overexpression does not have an effect on trichome initiation in either ga1-3 or gai. No increase in trichome density was noticeable on leaves, stems, or flowers of 35S:GIS gai plants, and carpels did not produce ectopic trichomes (Figure 3D). ga1-3 plants overexpressing GIS displayed a similar phenotype (see Supplemental Figure 3 online). To test whether GIS expression levels in transgenic plants were sufficient to induce the overexpression phenotype, we sprayed 35S:GIS ga1-3 plants with 100 µM GA. This treatment restored the overexpression phenotype normally seen in a wild-type background (data not shown). We also verified GIS expression levels in 35S:GIS gai and 35S:GIS ga1-3 transgenic lines and found that GIS was indeed overexpressed to significant levels (27- and 442-fold, respectively, in the lines we tested). The phenotype of GIS overexpressing gai and ga1-3 plants indicated that GAI-mediated repression of trichome initiation is independent of GIS expression levels. To test whether it was also independent of GL1 expression levels, we measured GL1 transcript levels in 35S:GIS gai, 35S:GIS ga1-3, and control plants. We found that GL1 was still repressed in spite of high levels of GIS expression in transgenic plants and in fact was expressed at similar levels in 35S:GIS gai, 35S:GIS ga1-3, and control plants (see Supplemental Figure 4 online). Therefore, repression of trichome initiation and GL1 expression in the absence of GA signaling, while it involves the GAI-mediated downregulation of GIS expression, occurs independently of GIS expression levels.
We report the identification of a putative C2H2 transcription factor, GIS, which regulates several aspects of shoot maturation in Arabidopsis. The analysis of overexpressors and loss-of-function mutants indicates that GIS plays a central role in the control of trichome initiation during inflorescence development. GIS also plays a role in the regulation of epidermal differentiation during the vegetative phase, although this function only becomes apparent when GA signaling is altered in the plant. The phenotype of gis spy double mutants indicates that GIS also modulates the repressive effect exerted by SPY on flowering. Through the analysis of gene expression profiles, genetic interactions, and effects of modulating GA levels in the plant, we have found that GIS acts downstream of the GA signaling pathway and controls epidermal differentiation by modulating the activity of one or more cognate regulators of trichome initiation.
Role of GIS in Shoot Maturation Our analysis of the genetic interactions between GIS, GL1, GL3, and TTG1 and of gene expression also suggests that GIS acts upstream of the trichome initiation complex and that it modulates the activity of the complex through a direct or indirect transcriptional mechanism. While the gain-of-function and loss-of-function phenotypes and gene expression data argue that GIS is an activator of inflorescence trichome initiation, the occasional appearance of rosettes on overexpressors could suggest that GIS is more generally repressing inflorescence shoot maturation and controls the timing of trichome initiation rather than initiation itself. This scenario could be supported by the broad expression of GIS during inflorescence development, which would be compatible with a regulatory role that goes beyond epidermal differentiation. Whether GIS plays a direct or indirect role in promoting trichome initiation should become clearer once its immediate targets are identified. Regardless of this mechanism, the residual production in gis of trichomes on early inflorescence organs where the gene is normally expressed suggests that other regulators act in parallel to control trichome initiation. The regulation of these redundant factors is also likely to be important to the progression of the epidermal differentiation program, and it will be interesting to determine whether they respond to distinct developmental signals. In this respect, it will be sensible to define the function of C2H2 proteins that are most closely related to GIS, as the similarity in sequence between the homologs is suggestive of redundancy.
The role of GIS in other aspects of shoot maturation appears to be more complex. The timings of vegetative phase change and flowering are not affected in the mutant under normal conditions, although the results of exogenous GAs and PAC applications imply that abaxial trichome initiation requires higher GA levels in the mutant than in wild-type plants. This observation is in agreement with the phenotype of gis spy double mutants and suggests that GIS plays a role in promoting abaxial trichome production during the juvenile–adult transition (which would support a role for GIS as an activator of trichome initiation throughout the plant). An interesting finding is that GIS loss of function also affects flowering time in the spy background. This effect appears to be independent of GA levels as GA- and PAC-treated gis mutants do not flower significantly earlier or later than wild-type plants. The two results are not necessarily inconsistent, as SPY has recently been shown to be implicated in the GA-independent control of flowering under long days through its interactions with GIGANTEA (Tseng et al., 2004 A positive role by GIS in the regulation of flowering under long days and in the regulation of vegetative phase change by GA is in apparent contradiction with the phenotype of GIS overexpressors. While in agreement during inflorescence development, the loss-of-function and gain-of-function phenotypes seem inconsistent during the vegetative phase, as both mutant and overexpressors are, under inductive conditions, delayed in phase transitions. One possibility is that overexpression of GIS results in a dominant-negative effect that equates loss of function in vegetative organs but not in inflorescence organs, for example, through the inhibition of a regulatory complex that only forms before flowering. Alternatively, aspects of the overexpression phenotype that are not mirrored in the mutant could be an artifact of ectopic expression. Regardless of what causes the preflowering phenotype of GIS overexpressors, it is clear that GIS is implicated in the control of phase change during vegetative development and acts by integrating GA-dependent and -independent signals. Identification of proteins interacting with GIS and functional characterization of close homologs should bring further definition to its mode of action during the vegetative phase.
GIS and GA Signaling In summary, GIS is required for the induction of trichome initiation through its role in regulating GL1 expression, but its downregulation in the absence of GA signaling is not necessary for the repression of trichome initiation and GL1 expression. These observations lead us to a model of GA control over trichome initiation, according to which GIS acts downstream of SPY but at the same step as GAI (and possibly other DELLA proteins) in the regulation of GL1 to influence trichome initiation (Figure 9 ).
In conclusion, the identification of GIS is a first step in the elucidation of mechanisms through which GAs control cellular differentiation and epidermal aspects of phase change. Further definition of these mechanisms and of the interaction between GA-dependent and -independent pathways is likely to come from continued analysis of the biological roles of GIS and other members of its clade.
Plant Material and Growth Conditions Arabidopsis thaliana ecotype Col-0 was used as a control for most experiments in this study. The gl1-1, ttg-1, gai-1, ga1-3, and spy-3 mutants were obtained from the Nottingham Arabidopsis Stock Centre, and the gl3-1 mutant was kindly proved by Alan Lloyd. The gis spy-3 double mutants were selected out of an F2 population by selection on Murashige and Skoog medium containing sulfadiazine and PAC. Double mutants were confirmed by selfing the selected F2s and ensuring all progenies were resistant to both sulfadiazine and PAC. Plants of the Lansdsberg erecta ecotype were grown as a control whenever necessary. In all trichome counting experiments, the plants were grown under a 16-h-light (90 µE·cm–2·s–2; 21°C) and 8-h-dark (18°C) cycle. Short-day experiments were performed with 8-h-light (90 µE·cm–2·s–2; 21°C) and 16-h-dark (18°C) cycles. Sepal trichome numbers were recorded  35 d after sowing, when the plant reached 11 cm in size, on three apical flowers per plant. Leaf trichomes were counted when the leaves had fully expanded, on a 1-cm–2 area in the middle of the leaf. When the main inflorescence stem reached 18 cm in size, the total number of trichomes was counted on the first two to three branches, and on one to three basal internodes, depending on the experiment. At least 20 plants were used for trichome count analysis for each of the treatment x genotype combinations. All the experiments were repeated at least once.
Isolation of a GIS Knockout Mutant
GA and PAC Applications for Sensitivity Assays and Gene Expression Analysis For experiments aimed at measuring induction of gene expression in response to GA, gai, gal-3, gis, and control plants were grown on soil until young inflorescence shoots or paraclades had reached a size of 2 to 3 cm. The plants were then sprayed with either 100 µM GA3 or a mock solution, and the shoots were harvested 4 or 6 h after treatment for RNA extraction.
Aqueous solutions of PAC (Sigma-Aldrich) were applied to soil-grown plants by soaking the pots for 2 d when the plants had reached the four-leaf stage, unless stem trichome density was to be measured, in which case PAC was applied at the eight-leaf stage. The PAC solution was then removed from trays, and normal watering was resumed. Trichome data were recorded
Molecular Biology
Cloning For all these cloning experiments, gene-specific fragments were first PCR amplified from cDNA (35S:GIS and GIS-RNAi) or genomic DNA (pGIS:GUS) using primers containing SalI and NotI restriction sites, purified using a gel extraction kit (Clontech) before restriction and cloning. The following primers were used: GIS overexpression, 5'-TTTCTCAGTCGACCGCCCAGTCTTTTTATCTCTC-3' and 5'-TCATTCAGCGGCCGCACACATCGTGCCGTTTCTT-3'; GIS-RNAi, 5'-CATTGTCGACTTACCGTCATTACCCGTCGT-3' and 5'-TCGCGGCCGCACACATCGTGCCGTTTCTT-3'. A 1.6-kb genomic fragment upstream of the start codon in GIS was amplified using the primers 5'-ATCTTGGTCGACTGCACACACTTTTATGGCAAA-3' and 5'-CTAATGGCGGCCGCGAGAGATAAAAAGACTGGGCG-3' for preparing the pGIS:GUS construct. The R gene was PCR amplified from maize cDNA using the primers 5'-CTGAGTCGACATCGAGTTGTTGTACTCTTCGCAGA-3' and 5'-TCGATCCCGCGGCCGCTTCCATGCCCGTCGATGTCCAAA-3', cloned first into the pMEN065 vector, then subcloned into the pBI101 vector.
All binary vector constructs were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. Agrobacterium-mediated transformation of all Arabidopsis genotypes was performed using the floral dip method (Clough and Bent, 1998
In Situ Hybridization
Phylogenetic Analysis
Accession Numbers
Supplemental Data
We thank Ottoline Leyser for useful comments on the manuscript, the Garfield Weston Foundation for funding Y.G.'s work, and all the scientists at Mendel Biotechnology who have contributed, directly or indirectly, to this work.
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.plantcell.org) is: Pierre Broun (pbroun{at}york.ac.uk).
[W] Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.106.041533. Received January 30, 2006; Revision received March 13, 2006. accepted March 30, 2006.
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INTRODUCTION
Ct) and then calculating 2–