INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Gibberellins (GAs) regulate many aspects of plant growth and development. In the seed and seedling these include the production of hydrolytic enzymes, germination, and growth. In the adult plant, GAs are important in leaf and stem elongation, flowering, anther development, and fruit set (Pharis and King, 1985).

Two classes of mutants have contributed much to an understanding of GA action (Thornton et al., 1999). One class includes dwarf mutants that are defective in GA biosynthesis. The other class includes response mutants such as Arabidopsis spindly (spy), GA-insensitive (gai), repressor of GA1-3 (rga), and the rice (Oryza sativa) d1 mutant. Many of the genes defined by these mutants have been cloned, but their molecular role in GA signaling is not yet fully understood (Jacobsen et al., 1996; Peng et al., 1997; Silverstone et al., 1998; Ashikari et al., 1999).

An alternative approach to understanding GA signal transduction has involved functional studies, particularly with aleurone cells of cereals. These studies have identified a number of early GA signaling steps that precede expression of hydrolytic enzymes such as -amylase. These steps involve heterotrimeric G-proteins (Jones et al., 1998; Ueguchi-Tanaka et al., 2000) and cGMP (Penson et al., 1996), which may in turn control the barley (Hordeum vulgare) HvGAMYB gene, whose expression is induced by GAs (Gubler et al., 1995).

HvGAMYB encodes a transcriptional activator that binds specifically to a GA-response element in an -amylase promoter (Gubler et al., 1995). Constitutive expression of HvGAMYB mimics the effects of GA application and is sufficient to activate the -amylase promoter and the promoters of other GA-regulated genes in aleurone tissue (Cercos et al., 1999; Gubler et al., 1999). However, GAMYB involvement in the response to GAs may not be restricted to aleurone. For example, during long day (LD)-induced flowering of the grass Lolium temulentum, GAMYB expression increases at the shoot apex (Gocal et al., 1999), shoot apex GA content increases at this time (King et al., 2001), and applied GA application mimics the effects of LD exposure on floral induction (Evans, 1964; Evans et al., 1990; King et al., 2001). Thus, we have proposed that GA, acting via GAMYB, activates genes that are responsible for floral initiation/development.

A potential target for GAMYB in transcriptional regulation of flowering is the LEAFY gene. LFY is a potent inducer of flowering in dicots, including Arabidopsis (Weigel and Nilsson, 1995), and can also accelerate flowering in a monocot, rice (He et al., 2000). The LFY gene is activated by application of GA (Blázquez et al., 1997, 1998) and the LFY promoter of at least two dicots contains a potential MYB-binding motif that is required for normal LFY promoter activity (Blázquez and Weigel, 2000). In addition, consistent with the role for GAMYB in regulating LFY, LtLFY is induced after LtGAMYB during the floral transition at the shoot apex of L. temulentum (Gocal et al., 1999, 2001).

Here, we describe three GAMYB-like genes from Arabidopsis, AtMYB33, AtMYB65, and AtMYB101, the proteins of which are capable of transactivating an -amylase promoter in barley aleurone cells. We show that AtMYB33 and AtMYB65 are co-expressed in many tissues, but AtMYB101 expression is restricted to the subapical pith cells of both vegetative and flowering plants and to the hypocotyl hook. We have used measurements of GA levels, together with manipulation of GA levels, to investigate the role of GA in regulating expression of this group of genes during elongation growth and flowering. At the shoot apex, the timing and pattern of expression of AtMYB33 precedes and overlaps with that of the LFY gene and, furthermore, AtMYB33 binds in vitro to a specific sequence in the LFY promoter. Such findings are compatible with a GA signaling role of GAMYB-like genes in flowering.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Three GAMYB-like Genes in Arabidopsis

Sequences of three Arabidopsis GAMYB-like genes (AtMYB33, AtMYB65, and AtMYB101) were obtained on the basis of sequence similarity to GAMYB genes from barley, rice, and L. temulentum (Gubler et al., 1995, 1997; Gocal et al., 1999). The nucleotide sequences of the three AtMYB cDNAs have been lodged with GenBank (accession nos. AF411969 for AtMYB33, AF048840 for AtMYB65, and AF411970 for AtMYB101). Based on cladistic analysis of partial sequences of more than 80 different AtMYB proteins, Kranz et al. (1998) defined a subgroup, number 18, which comprised these three genes, AtMYB33, AtMYB65, and AtMYB101, and a fourth, AtMYB81, which we have not studied. The graminaceous GAMYBs are most closely related to the four members of this Arabidopsis subgroup. All share a QRaGLPxYPx(E/S) motif (Fig. 1, Box 1) immediately C-terminal to the R2 R3 repeat DNA-binding domain (Kranz et al., 1998; Romero et al., 1998). AtMYB33 has the highest overall identity with HvGAMYB (41%) but, over the R2R3 repeat sequence alone, identity with HvGAMYB was 86.4%, 85.4%, and 82.5% for AtMYB33, AtMYB65, and AtMYB101, respectively. In addition, there are regions around amino acids 371 to 387 and at the C terminus that are conserved between cereal GAMYBs and AtMYB33, AtMYB65, and AtMYB101 (Fig. 1, Boxes 2 and 3). Other regions are conserved between a subset of the Arabidopsis genes and cereal GAMYBs (Fig. 1). The genomic structure of Arabidopsis GAMYB-like genes is also conserved with HvGAMYB (F. Gubler and R. Kalla, unpublished data), with the location of an intron at the 3' end of the open reading frame being unique to this class of MYB genes (Fig. 1). HvGAMYB has one intron in the 5'-untranslated region and AtMYB33 has two (G.F.W. Gocal, F. Gubler, R. Kalla, and C.C. Sheldon, unpublished data).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Comparison of the structure of the barley GAMYB protein (HvGAMYB) and the three Arabidopsis GAMYB-like proteins (AtMYB33, AtMYB65, and AtMYB101). Regions of similarity between all four proteins are shown in black, with the amino acid sequences of Boxes 1, 2 and 3 shown below. Regions of similarity between HvGAMYB and at least one Arabidopsis MYB are shown in gray. Similarity is defined as a continuous stretch of at least five amino acids with greater than or equal to 70% identity, or a continuous stretch of at least nine amino acids with greater than or equal to 50% identity. Amino acid numbers for the regions of similarity are shown for HvGAMYB. The location of the two introns present in the coding region of the genes is shown.

AtMYB33 is located on the top of chromosome 5 at approximately 1,840,000 bp, AtMYB65 on the top of chromosome 3 at approximately 3,610,000 bp, and AtMYB101 at approximately 14,065,000 bp, two-thirds of the way along chromosome 2.

Functional Analysis of the Three AtMYB Genes

In transient expression experiments, HvGAMYB can substitute for GA₃ in activating transcription of the GA-responsive -amylase promoters from barley (Gubler et al., 1995, 1999). We therefore determined whether this -amylase promoter could be activated by expression of AtMYB33, AtMYB65, and AtMYB101. Barley aleurone tissue was bombarded with a low-pI -amylase reporter along with each of the MYB effector plasmids. As a control for the specificity of GAMYB activity, we also assayed the ability of an unrelated Arabidopsis MYB gene, AtMYB2 (Urao et al., 1998), to activate the -amylase promoter. The -amylase promoter was strongly induced by AtMYB33, AtMYB65, and AtMYB101, but not by AtMYB2 (Fig. 2). The increase in GUS activity in response to AtMYB33, AtMYB65, and AtMYB101 was similar to that observed with HvGAMYB.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Transactivation of a barley -amylase promoter by Arabidopsis GAMYB-like proteins. Intact aleurone cells were cobombarded with a reporter construct containing an -amylase promoter fused to GUS and different Ubi1.MYB effector constructs: Ubi1.cas (a no-insert control), Ubi1.HvGAMYB, Ubi1.AtMYB33, Ubi1.AtMYB65, Ubi1.AtMYB101, and Ubi1.AtMYB2. The Ubi1.cas expression cassette is described by Gubler et al. (1999) The bars are twice the SE of the mean for the eight to 16 replicates per assay.

Expression Patterns of Arabidopsis GAMYB-like Genes

We used RNase protection to analyze the tissue-specific expression pattern of the AtMYB genes in the Columbia ecotype (Fig. 3). The expression of AtMYB65 was approximately 10-fold less than that of AtMYB33 or AtMYB101. AtMYB33 was expressed in all tissues, but it also showed a predominantly floral expression pattern. AtMYB65 expression was fairly similar across all tissues although with a slight increase in floral tissues.

View larger version (49K): [in this window] [in a new window]   Figure 3.   Expression pattern of Arabidopsis GAMYB-like genes, AtMYB33, AtMYB65, and AtMYB101, as determined by RNase protection assays of mRNA levels in various tissues from Columbia plants. The negative control for the RNase protection assay contained only yeast tRNA. The floral shoot tip included flower clusters at the tip of the inflorescence but with no visible petals.

The expression patterns of the three genes were examined in more detail by in situ hybridization. In germinating seeds, AtMYB33 RNA was found in the root tip and in a linear array of up to 20 to 30 cells above the root tip (Fig. 4 A). The vegetative shoot apex showed weak expression of AtMYB33 both in germinating seeds and 60-d-old plants held in SDs (Fig. 4, C and E). Higher levels of expression were evident in primordial leaves which probably include developing petioles (Fig. 4E). As a control for specificity, there was no hybridization of AtMYB33 sense probes as shown in Figure 4, B and G. 

View larger version (188K): [in this window] [in a new window]   Figure 4.   Localization by in situ hybridization of expression in Arabidopsis of GAMYB-like gene transcripts of AtMYB33, AtMYB65, and AtMYB101. Germinating seeds (A-D, Columbia wild type) were harvested 48 h after imbibition. Shoot samples were from vegetative (E and H) or florally induced (F, G, I, and L) plants. The shoot tissue was either from Columbia (H, J, and L) or Col hy4-101 (E, F, G, I, and K), however, there were no differences between the genotypes in expression patterns in specific tissues (not shown). Control sections hybridized with AtMYB33 sense probes are shown in B and G. Probe concentrations, DIG antibody concentration, and duration of color development were identical in all panels. Bars = 100 µm. am, Apical meristem; fl, flower primordia; lo, developing stamen locule; o, ovule; rm, root meristem.

Inflorescence and flower primordia had developed to floral stages 2 to 3 (Smyth et al., 1990) at 6 d after transfer of plants to LDs. Compared with the vegetative shoot apex, AtMYB33 was strongly expressed in the inflorescence apex (Fig. 4F). Its expression was comparatively weaker in the inflorescence stem, the vascular tissue, and the vascular tissue in leaf primordia. In the gynoecium only ovules showed expression (Fig. 4J), and in developing anthers AtMYB33 was expressed in developing locules of immature anthers and later, albeit weakly, in pollen grains (Fig. 4, I and J). The expression pattern of AtMYB65 in the root apex (not shown) and vascular strands and floral apices of the developing inflorescence (Fig. 4K) paralleled that of AtMYB33.

The pattern of AtMYB101 expression was very different from that of AtMYB33 and AtMYB65 (Fig. 3). There was little or no expression of AtMYB101 in the root tip of germinating seedlings (Fig. 4D), in the vegetative (Fig. 4D), early floral (Fig. 4H), or inflorescence shoot apex (Fig. 4L), or in immature anther and carpel tissue (not shown). Expression of AtMYB101 was detected in a small patch of cells on the innermost side of the hypocotyl hook of the germinating seedling (Fig. 4D), in the subapical pith cells of plants growing vegetatively (Fig. 4H), in a similar zone of expanding cells both in developing inflorescence stems (Fig. 4L), and below mature flowers and elongating siliques (not shown).

Effects of GAs on Petiole Elongation

In plants such as spinach, exposure to LDs enhances growth and erectness of petioles, and there is an accompanying increase in GAs (Zeevaart, 1971; Talón et al., 1991; Zeevaart and Gage, 1993). Here we show comparable responses in Arabidopsis associated with LD exposure, which caused young petioles to grow faster and to become more erect. These responses occurred soon (within 1-2 d) after transfer of plants of Col hy4-101 from SDs to LDs, their length at 6 d being about 1.5 times that of plants kept in SDs (Fig. 5A). The angle of the petiole to the horizontal almost doubled over the same 6-d period (12.5 ± 0.5° to 20.5 ± 0.8°).

View larger version (19K): [in this window] [in a new window]   Figure 5.   Arabidopsis petiole elongation in response to exposure to LDs or GA (millimeter increase over the starting length). A, control plants in SDs (about 60-d old plants of Col hy4-101; ); transferred to LDs at the start of treatment (); in SDs but treated once with GA4 (15 µg in water applied to the shoot tip; ). B and C, Application in SDs of GA4 (; 66 ng in 95% ethanol applied to the measured petiole); Paclobutrazol (; abbreviated as PP333), a GA biosynthesis inhibitor (200 ng in 95% ethanol), or 66 ng of GA4 plus 200 ng of paclobutrazol (). Control plants were treated with water (A) or 95% ethanol (B and C). The dotted line in C is the relevant control from B. Bars are the value of the LSD at P = 0.05. There were eight replicate plants per measurement.

In three ways these findings suggest that GA mediates the LD growth response. First, a single application of GA to SD-grown plants mimicked the effect of LDs on petiole elongation (Fig. 5). The elongation response differed between the SD controls in the two experiments shown (A versus B) but GA response equal to the LD response was found in both experiments (LD not shown for the experiment in Fig. 5B). Second, blocking GA synthesis with paclobutrazol inhibited the LD increase in petiole length, the increment at 3 d being 4.5 ± 0.7 mm in LD, 2.1 ± 0.2 mm with paclobutrazol application in LD, and 2.5 ± 0.4 mm in SD. Such effects of the chemical were specific because, in SDs, the 40% reduction in petiole elongation by paclobutrazol was reversed by application of GA₄ (Fig. 5C). Third, in the same experiments, LD-induced elongation of young petioles was matched by substantial increases in petiole GA content as shown in Figure 6 and Table I.

View larger version (16K): [in this window] [in a new window]   Figure 6.   Increase in the endogenous GA content of young petioles after exposure of the plant to LDs. Young elongating petioles were harvested from SD plants () or at various times after exposure to LD () as part of the experiment detailed in Figures 4, 5A, and 7. Data for all 10 GAs assayed in this experiment is shown in Table I.

The content of bioactive GA₁ and GA₄ increased within 2 d of transfer to LDs, and by 6 d had increased 30-fold for GA₁ and 6-fold for GA₄ (Fig. 6; Table I). These findings with single assays were confirmed in a second, more restricted analysis in which, after two LDs, the increase in GA₁ was 2.8-fold (Table I). The levels of GA₄ were comparable between the two experiments, however, for plants in SDs more GA₁ was detected in the second than the first experiment (i.e. 0.045 and 0.052 ng g¹ dry weight versus 0.013 ng g¹ dry weight; compare with Table I).

There were no large changes in precursors of GA₁ such as GA₅₃, GA₄₄, GA₁₉, or GA₂₀ although two immediate precursors of GA₄, GA₂₄ and GA₉, increased somewhat (Table I). Part of the GA₁ increase could reflect a reduced catabolism to GA₈ (Table I). However, the drop in the level of GA₈ in LDs was not reproduced in the second experiment, a finding we cannot explain. Interestingly, compared with young petioles, no increase in GA levels could be detected in extracts of shoot tissue including leaves and old expanded petioles (data not shown), and this probably explains why Xu et al. (1997) could not detect any dramatic increase in LDs in GA₁ and GA₄ levels in their analysis of total shoot tissues of Arabidopsis.

Effects of LD and GAs on Flowering and AtGAMYB Expression

GA can enhance flowering of Arabidopsis and may be essential in SDs as demonstrated by Wilson et al. (1992) with a GA-deficient mutant. Furthermore, as shown in Table II, in the experimental conditions used here, flowering of Col hy4-101 plants was induced equally well by a single application of GA₄ in SDs or by exposure to a single LD. However, such information on flowering is not definitive despite the parallel effects of GA on petiole elongation and on endogenous GA levels (Figs. 5 and 6). For flowering, we require information on shoot apex GA content but this is a challenging task because of the extremely small size of the shoot apex.

In the same set of plants used above for petiole growth and flowering, expression of the GA-responsive floral meristem-identity gene LFY was very low in the vegetative apex but increased dramatically after the 2nd d of exposure to LDs, i.e. during early inflorescence differentiation (Fig. 7, A-E). A comparable increase over time in expression of AtMYB33 was evident during floral differentiation (Fig. 7, F-J). Apex height provided a more sensitive marker of floral response than the morphological floral stage shown in Figure 7. Apex height had increased after 2 d and probably before then (Fig. 7K). At these early times it was the apex alone that increased in height. Later (at 6 d) the new stem made up a larger component of the "apex" but all tissue was derived from the initially rather flat apex (Fig. 7, A, F, and K) rather than by activation of any preexisting subapical meristem, a finding quite unlike previous reports for other species (Sachs, 1965).

View larger version (50K): [in this window] [in a new window]   Figure 7.   Increase at the shoot apex in expression of AtMYB33 and LFY mRNA and change in shoot apex morphology over the early days of LD-induced flowering of Arabidopsis. Expression of LFY (A-E) and AtMYB33 (F-J) was analyzed by in situ hybridization. Median longitudinal shoot apex sections are shown for: vegetative 60-d-old Col hy4-101 plants growing in SDs (A and F); or during the floral transition after exposure to 24-h LDs for 2 d (B and G), 4 d (C and H), or 6 d (D and I). LFY expression at a later stage of floral development is shown in E. A sense control for AtMYB33 is shown in J after 4 LDs. Floral scores are based on the scale proposed by Smyth et al. (1990). Change in shoot apex morphology during floral induction (shown in K) was determined by measuring shoot apex height (in micrometers) above a line joining the insertion point of the last two leaf primordia. The averages are based on the seven separate apices sectioned for AtMYB33 mRNA localization. Bars indicate two times SE. The plants exposed to LD were part of the experiment shown in Figures 4 and 5.

Application of GA₄ to plants in SDs induced flowering in our experiments (Table II), and AtMYB33 expression increased with inflorescence differentiation (Fig. 8) in a parallel manner to that seen in Figure 7 for LD induction of flowering. Expression of AtMYB33 in the immature anther locule (Fig. 4I) was evident also after GA application (C.P. MacMillan and R.W. King, data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 8.   Increase in expression at the shoot apex of AtMYB33 with GA4 induced flowering of plants in SDs. Sixty-day-old plants were treated twice over 4 d with GA4 (66 ng plant1). Expression of AtMYB33 is shown at the start of treatment (A), at 8 d (B), or 12 d (C) and in D for a sense probe of an apex harvested 8 d after the start of treatment. Floral scores are based on the scale proposed by Smyth et al. (1990).

One potential target in Arabidopsis for GAMYB action is the LFY promoter. Functional analysis of the LFY promoter has identified a cis-acting element critical for GA responsiveness (Blázquez and Weigel, 2000). Removal of this element within the context of a synthetic LFY promoter, referred to as GOF9, renders this LFY promoter insensitive to GA₃ and reduces its activity particularly in SDs. Within this cis-acting element there is a motif CAACTGTC (approximately 249 to approximately 242), which is a putative GAMYB-binding site based on the deduced consensus-binding site of GAMYB, which is ^C/_TAAC^C/_G^G/_A^A/_C^C/_A (Gubler et al., 1999).

To examine the specificity of the LFY promoter GAMYB-binding motif, the AtMYB33 protein was expressed in E. coli and tested for its ability to bind to a 287-bp fragment from the LFY promoter (position 375 to 88), which includes the putative MYB-binding site. AtMYB33 bound strongly to the 287 base promoter fragment causing a shift in electrophoretic mobility (Fig. 9). When six of the eight bases in the putative binding site were mutated, binding of AtMYB33 was reduced by more than 75% (Fig. 9).

View larger version (44K): [in this window] [in a new window]   Figure 9.   In vitro binding of AtMYB33 to the LFY promoter. AtMYB33 protein binding in vitro to a 287-bp 32P-labeled fragment of the LFY promoter as seen by an electrophoretic mobility shift on a nondenaturing polyacrylamide gel. A 6-bp change (underlined) in the DNA sequence (pGG9) disrupts a putative GAMYB-binding site and reduces DNA binding by 75%.

Blázquez and Weigel (2000) reported that the same six base mutations in the context of the LFY GOF9 promoter caused a selective reduction of LFY promoter activity in SDs and removal of GA responsiveness. Taken together, these results suggest that AtMYB33 might influence flowering by mediating GA responsiveness of the LFY promoter.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

A Group of Three GAMYB Genes in Arabidopsis

GAs regulate many developmental processes in plants, but only a few downstream effectors have been identified. One of these is the GAMYB transcription factor from barley. Here we have shown that the protein products of three GAMYB-related genes from Arabidopsis, AtMYB33 AtMYB65, and AtMYB101, are likely to transduce GA signals in this species.

Structurally, these three GAMYB-like proteins of Arabidopsis constitute a distinct subgroup that clusters most closely with GAMYBs from barley and other Gramineae and less so with other Arabidopsis MYBs containing an R2R3 DNA-binding domain (Gubler et al., 1995, 1997, 1999; Kranz et al., 1998; Gocal et al., 1999). Although the GAMYB clade in Arabidopsis has several members, to date, only one GAMYB-related gene has been found in each monocot species. Functionally, all the Arabidopsis genes readily substitute for the barley GAMYB in their transactivation of the barley -amylase promoter (Fig. 2). By contrast, two structurally distinct MYB genes were inactive, AtMYB2 (Fig. 2) and C1 (Gubler et al., 1995).

AtMYB33 and AtMYB65 may constitute a redundant gene pair with common functions in the plant as suggested by their similar expression patterns (Fig. 4). The divergent expression pattern of AtMYB101 suggests it plays a different role(s). Because of expression of AtMYB101 in subapical tissue but not in the shoot or root apex, we speculate that it may be involved in GA-regulated stem elongation. This would fit with the finding that GA application causes stem elongation (bolting) in association with enhanced cell division in subapical tissue in a number of rosette plants (Sachs, 1965) including Arabidopsis (Besnard-Wibaut, 1970).

GAs and Flowering

GAs play an important role in mediating the flowering responses of several LD plants. One particularly well-studied example is with the LD and GA-responsive grass, L. temulentum (Evans et al., 1990; King et al., 1993). The content of GA₁ and GA₄ in L. temulentum leaves (Gocal et al., 1999) and the shoot apex (King et al., 2001) increases with exposure to two or more LDs, and there is a related up-regulated expression of LtGAMYB at the shoot apex (Gocal et al., 1999). Thus, these endogenous GAs may regulate the GAMYB expression seen at inflorescence formation. Similarly, GAs may be important for flowering of Arabidopsis because the GA-deficient mutant ga1-3 only flowers in SDs if GA is applied (Wilson et al., 1992). In LDs, GAs are still important but to a lesser extent because there is still a significant delay in flowering of the ga1-3 mutant (Wilson et al., 1992), and LD responsiveness of the LFY promoter is attenuated in this mutant (Blázquez et al., 1998). Here we have provided more certain evidence of a link between flowering, GA content, and changes in downstream molecular events.

Our claim of a link between GA and flowering is based principally on five observations: (a) A single GA₄ application induced flowering in SDs (Table II). (b) When LDs caused flowering (Fig. 7) there was a simultaneous increase in endogenous GA₁ and GA₄ in the petiole (Table I; Fig. 6) and, we assume, also in the shoot apex. (c) In association with flowering LD exposure led to enhanced expression at the shoot apex of LFY and AtMYB33 (Fig. 7). (d) When GA₄ was applied, it induced expression of AtMYB33 at the shoot apex in association with flowering (Fig. 8). (e) The promoter of the LFY gene contains an AtMYB33-binding motif (Fig. 9), which therefore provides a basis for linking GA to LFY expression.

There was an immediacy in these responses to LDs in that changes were evident over the first 2 d of floral induction. Simultaneously, there were increases in the size of the prefloral shoot apex (Fig. 7), in endogenous GA content of petioles (Fig. 6), and in petiole length and erectness (Fig. 5). Presumably, LDs caused GA content to increase in the shoot apex as rapidly as in the young petiole, a presumption supported by the evidence of increased expression of AtMYB33, which is apparently a GA-regulated change (e.g. Fig. 8). We also believe that a LD-induced increase in GA content of the Arabidopsis shoot apex is likely given our recent finding of changes in GA content of the shoot apex of L. temulentum after exposure to LD(s) (King et al., 2001). Previous studies of effects of daylength on Arabidopsis have shown little or no change in GA content due to LDs, but this was for whole shoots (Xu et al., 1997), a tissue mix in which we also found no detectable change in GA content (R.W. King, unpublished data).

Our findings are problematic when considering the studies with a GA biosynthetic mutant by Wilson et al. (1992) that were recently confirmed by Reeves and Coupland (2001). On the one hand, these genetic studies imply regulation of flowering of Arabidopsis by a LD-independent, GA-dependent pathway and, separately, by a LD-dependent, GA-independent pathway (Piñeiro and Coupland, 1998). On the other hand, our studies here with Arabidopsis and earlier with the LD-responsive grass L. temulentum (King et al., 2001), strongly link flowering, LD exposure, and GAs. Possibly some of the reports of LD-dependent, GA-independent flowering (Blázquez et al., 1998; Piñeiro and Coupland, 1998, and refs. therein; Blázquez and Weigel, 2000) can be explained as photosynthetic effects of LDs, a suggestion supported by our recent evidence that photosynthetic Suc input limits flowering in phyA mutants of Arabidopsis (Bagnall and King, 2001). Nevertheless, where low-irradiance LD photoperiods are employed as were here or in the studies of Wilson et al. (1992) and Reeves and Coupland (2001), there is no likelihood of a LD increase in photosynthetic input. Perhaps, therefore, in LDs there are both GA-dependent (our study) and GA-independent (Wilson et al., 1992; Reeves and Coupland, 2001) responses, with photosynthetic input being a further component of the LD, GA-independent pathway(s).

As an aside, it is well known that GA regulates petiole elongation and plant habit in a number of plant species and, as shown here, also in Arabidopsis. Such change in morphology induced by GA has been particularly well documented for spinach (Zeevaart, 1971; Metzger and Zeevaart, 1985; Talón et al., 1991). There may be a GAMYB signaling role in such elongation growth in Arabidopsis, but we were unable to detect in petioles any change in expression of the three AtMYB genes during GA or LD-enhanced elongation (C.C. Sheldon, unpublished data). Similarly, although the differences in tissue specific expression patterns shown in Figure 4 might suggest roles for AtMYB33 and AtMYB65 in GA regulation of germination, testing this possibility has been difficult, particularly in the absence of mutants.

Another issue raised by our study relates to the nature of the first events of flowering. Despite the evidence of a link between applied GA and expression of the floral regulatory gene, LFY (Blázquez et al., 1998; Blázquez and Weigel, 2000), and of AtMYB33, it must be questioned whether LFY or AtMYB33 regulate any of the events during the first 2 LDs. In our studies, expression of these two genes had barely increased after 2 LDs when the apex was already progressing to flowering as shown by its increase in size (Fig. 7). Perhaps, for Arabidopsis, LFY and GAMYB expression is more associated with later inflorescence formation, as also reported in earlier studies with L. temulentum (Gocal et al., 1999, 2001). In previous work with Arabidopsis, LFY was expressed early during floral development and even in the shoot apex of vegetative plants (Blázquez et al., 1997; Hempel et al., 1997). However, compared with the domed apex of the "vegetative" plants used in earlier studies (e.g. Blázquez et al., 1997; Hempel et al., 1997), our vegetative plants had a very flat apex that was essentially devoid of LFY expression (e.g. Figs. 4, 7, and 8). Thus, it is possible that in these earlier studies, the shoot apex had already progressed developmentally beyond the earliest events of "floral evocation."

Expression of LFY during the inflorescence transition implies late GA-regulation but does not exclude a separate, earlier action of GAs on flowering. In this context, our recent measurements of the content of GAs at the shoot apex of L. temulentum (King et al., 2001) highlight two such GA actions during flowering. Following the 1st d of LD exposure (i.e. at floral evocation of L. temulentum), the GA₅ content of the apex doubled, after which expression increased for two floral-specific APETALA1-related genes (Gocal et al., 2001). A few days later at inflorescence initiation, there were large increases in the content of bioactive GA₁ and GA₄ (King et al., 2001), after which there was increased expression of LtGAMYB (Gocal et al., 1999) and of LtLFY (Gocal et al., 2001). Thus, as for L. temulentum, our evidence with Arabidopsis is consistent with applied or endogenous GAs acting as regulators of LFY expression and, hence, of flowering, but increased LFY expression is not necessarily the earliest "floral" response to GAs.

GAs, Flowering, and a GAMYB/LEAFY Signaling Pathway in Arabidopsis

The scenario of GA regulating flowering via its activation of LFY gene expression raises the possibility that transcriptional regulators involved in the GA signal transduction pathway might be regulators of LFY. AtMYB33 is a candidate for such a regulator. Not only could it replace GA in activating the -amylase promoter in transient expression assays (Fig. 2), but also its expression increased at the shoot apex (Fig. 7, 8) in association with GA application (Fig. 8) or with increased endogenous GA levels in the plant (Fig. 6). However, most cogently, we have identified a potential regulatory hierarchy involving AtMYB33 binding to a GA-responsive region of the promoter of the floral meristem-identity gene LFY (Fig. 9). Recent studies of Blázquez and Weigel (2000) provide further support for such a signaling hierarchy and for in planta relevance of our studies. They found that the same mutation in the LFY promoter that we used to block AtMYB33 binding also blocks GA-induced expression of a LFY-GUS reporter gene construct.

Overall, our studies extend our earlier evidence (Gocal et al., 1999) that the action of GA on flowering may be via the GAMYB class of transcriptional regulators and, potentially, via transactivation of a specific floral regulatory gene, LFY.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material, Growing Conditions, and Chemical Treatments

Plants of Arabidopsis ecotype Columbia wild-type or a blue-photoreceptor (cryptochrome 1) mutant (Col hy4-101) were grown in soil in an 8-h short day (SD) at an irradiance of 100 µmol photons m² s¹ at 22°C. In these conditions involving exposure to blue-rich fluorescent tubes, hy4-101 plants took up to 26 weeks to flower. However, for flowering its phytochrome responses are unaltered compared with Columbia (Bagnall et al., 1996). Thus, in the experiments here (see below), we could induce flowering within 1 to 2 d on transferring 8-week-old plants to 24-h LDs given as a 16-h extension at low irradiance (10 µmol photons m² s¹) from far-red-rich incandescent lamps. Such a combination of genetics and photophysiology with its focus on LD phytochrome inputs also avoided the often substantial LD photosynthetic effects on flowering that we have recently documented in Arabidopsis (Bagnall and King, 2001).

GAs were synthesized and supplied by Prof. Lewis. N. Mander (Research School of Chemistry, Australian National University, Canberra, Australia). The GA biosynthesis inhibitor, paclobutrazol ([2S,3S]-1-[4-chlorophenyl]-4,4-dimethyl-2-[1,2,4-triazol-l-yl] pentan-3-ol), was obtained as the pure enantiomer from Dr. J. Lenton (Long Ashton Research Station, UK). Chemicals were applied directly to shoot tips in 40 µL of aqueous solution and included 5% ethanol and 0.1% Tween 20 as a wetting agent. Alternatively, chemicals were applied directly to young petioles as a 2-µL drop in 95% ethanol. Controls were treated with the same solution without the test chemical.

Isolation of cDNA and Genomic Clones

Cloning of AtMYB33

Cloning of AtMYB65

Cloning of AtMYB101

RNase Protection

Total RNA was extracted from approximately 0.5 g of plant tissue, following the method of Logemann et al. (1987). Templates for RNA probe preparation were generated by PCR amplification of the 3' region of each gene, containing part of the 3' intron and the 3' coding region. The primers were: AtMYB33F(BamHI), 5'-CGCGGATCCACACAAAATGCAGATG-3'; AtMYB33R(KpnI), 5'-CGGGGTACCAATGGAGTGGAGGAT-3'; AtMYB65F(BamHI), 5'-CGCGGATCCCTCGCAACTTAGTGC-3'; AtMYB65R(KpnI), 5'-ACGGGGTACCGTTACAGCGACCAAACAG-3'; AtMYB101F(BamHI), 5'-CGCGGATCCTTCTCATCATTCATCATTG-3'; and AtMYB101R(SalI), 5'-ACGCGTCGACGTTCCAAACTAACAGATGC-3'.

The PCR products were digested with the restriction enzymes indicated in the primer name (restriction sites indicated in bold, annealing sequence underlined) and cloned into the appropriately digested pBluescript SK and KS (Stratagene, La Jolla, CA). Constructs were sequenced to ensure their identity. RNA probes were synthesized using a BamHI-linearized pBluescript SK-AtMYB33, 65 and 101 plasmid, for antisense transcript probe or a KpnI-linearized pBluescript KS-ATMYB33 for a sense transcript probe and T7 RNA polymerase (Promega, Madison, WI) according to the manufacturer's protocol.

RNase protection assays were carried out using the Hybspeed RPA kit (Ambion, Austin, TX) with 10⁵ cpm of the appropriate riboprobe and 20 µg of total plant RNA following the manufacturer's protocol. Protected fragments were precipitated, separated on 5% polyacrylamide/8 M urea gels and analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The assays were run three times and gave similar results each time.

In Situ RNA Localization

Seeds of Columbia were imbibed on water-soaked filter paper in the dark at 4°C for 2 d and then germinated at 22°C under continuous light. After 48 h the radicle first protruded, and seeds were then fixed for sectioning (Ferrándiz et al., 2000). For vegetative and floral shoot apices, 60-d-old plants of Col hy4-101 were induced to flower by transfer from SD for between 1 and up to 6 LDs. On longitudinal sections of the shoot apex, the first signs of transition to flowering were evident, microscopically, after as few as 2 LDs. Maturing pollen-stage flowers were obtained from Columbia wild-type and hy4-101 plants. In situ hybridization was as described by Ferrándiz et al. (2000). For the Arabidopsis LFY gene the probe pDW119 was used (Weigel et al., 1992). For the probes for each of the three AtMYB genes a 3' non-cross-hybridizing region of each gene was generated as detailed below.

An AtMYB33-specific EcoRI/SmaI fragment was subcloned into the EcoRI/EcoRV sites of pBluescript SK⁺ and KS⁺. A 450-bp region of the 3' AtMYB65 cDNA sequence was amplified by PCR with primers 5'-GGACTAGTGCAGGGAATGTTGTAAAG-3' and 5'-CCGCTCGAGTATATATAAATGCCTTCA-3' (restriction enzyme sites in bold, annealing sequences underlined) using the expressed sequence tag 166K23 as template. The PCR product was digested with XhoI and SpeI and cloned into XhoI and SpeI digested pBluescript SK and KS.

A 330-bp region of the 3' AtMYB101 cDNA sequence was amplified by PCR with the primers; 5'-AGTGGAATTCTTATGGGAAACC-3' and 5'ACGGGGTACCTCATTCCTCATCTCTTTCA-3' (restriction enzyme sites in bold, annealing sequences underlined) using the AtMYB101 cDNA clone as template. The PCR product was digested with EcoRI and KpnI and cloned into EcoRI- and KpnI-digested pBluescript SK and KS. Sense and antisense DIG-labeled in vitro-transcribed riboprobes were synthesized from the T7 promoter of AtMYB33 subclones linearized with EcoRI, AtMYB65 subclones linearized with XhoI and SpeI, respectively, or AtMYB101 3' subclones linearized with KpnI and EcoRI, respectively

DNA Constructs for Transient Expression Assays

The maize (Zea mays) ubiquitin gene expression cassette, Ubi1.cas, and Ubi1.HvGAMYB have been previously described (Gubler et al., 1999). PCR-generated fragments were inserted into Ubi1.cas to create Ubi1.AtMYB33 and Ubi1.AtMYB101. The HincII/SmaI fragment of the full-length AtMYB65 cDNA was inserted into the SmaI site of pUbi1.cas resulting in Ubi1.AtMYB65. An AtMYB2 cDNA (a gift from R. Dolferus, Commonwealth Scientific and Industrial Research Organization, Canberra, Australia) was cloned into Ubi1.cas to generate Ubi1.AtMYB2. The mlo22 construct containing the barley low-pI -amylase promoter -glucuronidase (GUS) reporter has been described (Lanahan et al., 1992).

Transient Expression Analyses

For cobombardment experiments, 1 µg of effector and 2 µg of reporter plasmid were precipitated onto 3 mg of gold as described (Gubler et al., 1999). Following bombardment of barley half-grains (Hordeum vulgare L. cv Himalaya), they were incubated in 2 mL of medium without hormone (10 mM CaCl₂, 150 µg mL¹ cefotaxime, and 50 IU mL¹ nystatin). After 24 h at 25°C, the bombarded half grains were frozen and stored at 20°C. Extract preparation and GUS activity assays have been described (Lanahan et al., 1992).

In Vitro DNA Binding

AtMYB33 protein with an amino-terminal GST and carboxy-terminal hexa-His was expressed in Escherichia coli BL21-DE3 cells and affinity-purified over a nickel-nitrilotriacetic acid agarose column (Qiagen, Valencia, CA). This expression construct (pGG19) was a derivative of pGEX4T3 (Pharmacia, Piscataway, NJ). The region between 375 and 88 of the LFY promoter was subcloned between the PstI and XhoI sites of pBluescript KS⁺ to create pGG10. A mutated construct, pGG9, contains the same region as in pGG10, but has the MYB-binding site mutation (GTCCatcgatTCAATTT) found in GOF9 m (Blázquez and Weigel, 2000). Inserts between NotI and XhoI from pGG9 and pGG10 were gel purified and end filled with [³²P]dCTP using Klenow. In vitro DNA binding was carried out as described by Parcy et al. (1998).

Analysis of Endogenous GA Content

Methods for GA extraction were as outlined by Gocal et al. (1999). The smallest samples, young petioles, had dry weights of 0.1 to 0.5 g. Dideuterated internal standards were supplied by Prof. L. N. Mander (Research School of Chemistry, Australian National University, Canberra, Australia). After HPLC and methylation, the purified samples were passed through an amino column, silylated, and analyzed by high-resolution gas chromatography-mass spectrometry with selected ion monitoring (Moritz and Jensen, 1995; King et al., 2001).