MAX2 is expressed throughout the plant
MAX2 transcript was detected by RT-PCR in all plant organs tested, including cauline and rosette axillary buds of plants at early reproductive stage (Figure 1a). To investigate tissue and subcellular localization of the MAX2 protein, 3.45 kb of MAX2 upstream sequence and the MAX2 coding region, which lacks introns, were fused to the GUS reporter gene. This construct, M2p::M2–GUS, partially complemented the highly branched max2 mutant phenotype. Out of five homozygous, single-insert lines in the max2 background, which showed similar GUS staining patterns, we chose the best-complemented line (Figure 1b) for detailed study. GUS activity was detected in many cell types, but was highest in the vasculature of growing leaves, flowers, siliques and stems (Figure 1c–f). In siliques, the funiculi, which connect the developing seeds to the placenta, stained particularly strongly (Figure 1e). In transverse sections of the vasculature of elongating inflorescence stems, GUS was detected in phloem, cambium and xylem parenchyma cells (Figure 1f). Vascular GUS activity declined when leaves and stems ceased growing, but remained high in the funiculi of ripening siliques (data not shown). Buds developing in rosette leaf axils (Figure 1g) showed uniform GUS staining. In the root, GUS activity was highest in developing vascular, pericycle and endodermal cells at the tip; weak staining was also detected in outer cell types and the root cap (Figure 1h,i). The overall vascular staining intensity decreased towards the base of the root (compare Figure 1i,j; root tip versus root hair differentiation zone). The subcellular pattern of GUS activity, for example in differentiating root cells (Figure 1i,j) and leaf trichomes (Figure 1k), suggests that the MAX2–GUS fusion concentrates in the nucleus. Nuclear accumulation of MAX2–GUS fusion protein was confirmed for dark-grown hypocotyl cells by comparing the patterns of GUS activity and of nuclear staining with the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI). In hypocotyls expressing a control construct, a fusion of amino acids 1–26 of MAX2 with GUS expressed from the MAX2 promoter, we observed diffuse GUS staining which differed from the DAPI staining pattern (Figure 2a,b). In contrast, hypocotyls expressing the full-length MAX2–GUS fusion under the MAX2 promoter, showed dots of GUS activity which co-localized with DAPI fluorescence (Figure 2c,d).
Figure 1. MAX2 is expressed throughout the plant. (a) Detection of MAX2 transcript by RT-PCR in total RNA from different organs. Seedlings were harvested after 13 days of growth on vertical agar plates. Vegetative shoot tissues were harvested from soil-grown plants, either when the primary inflorescence was 3–4 cm high or when fully elongated (labelled ‘late’). Flowers were harvested at stage 14 (Smyth et al., 1990) and siliques 4 days past stage 14. Detection of ACTIN2 transcript was used as a cDNA normalization control. cyc, cycle number. (b) Partial rescue of the max2 branching phenotype in a transgenic max2 line expressing MAX2–GUS fusion protein from the MAX2 promoter (M2p::M2–GUS). Branching was assessed in a decapitation assay (see Experimental procedures). The mean number of rosette branches of at least 2 cm length 10 days after decapitation is shown (error bar = SEM, n = 20). (c–k) Different organs of M2p::M2–GUS (max2) plants stained for GUS activity. If not mentioned otherwise, harvest stages were as in (a): (c) top rosette leaf (bar = 5 mm); (d) flower (bar = 1 mm); (e) silique (bar = 1 mm); (f) stem (part of a transverse section of the second internode of the primary inflorescence, with a vascular bundle in the centre, bar = 50 μm); (g) rosette axillary bud from a plant induced to flower, prior to bolting (bar = 100 μm); (h) primary root, harvested after 5 days of growth on vertical agar plates (transverse section about 0.4 mm above the root tip, bar = 50 μm); (i) root tip and (j) root hair differentiation zone in longitudinal sections of the primary root, harvested after 7 days of growth on vertical agar plates (bars = 50 μm); (k) leaf trichome (bar = 50 μm).
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Figure 2. MAX2–GUS fusion protein is nuclear-localized in dark-grown hypocotyl cells. Fixed 5-mm segments from 5-day-old dark-grown hypocotyls were stained for GUS activity, and with DAPI to detect nuclei. The figure shows about one-third of the width of the hypocotyls, with the outside on the left. Scale bars = 50 μm. (a, b) Transgenic control line expressing GUS fused to the 3.45-kb MAX2 upstream sequence plus the sequence encoding N-terminal amino acids 1–26 of the MAX2 protein (M2p::M2(1–26)–GUS) in the wild-type background. GUS activity (a, brightfield illumination) and nuclear staining (b, epifluorescence illumination) do not co-localise. (c, d) Transgenic line expressing GUS fused to the 3.45-kb MAX2 upstream sequence plus the complete MAX2 coding sequence (M2p::M2–GUS) in the wild-type background. GUS activity (c, brightfield) and nuclear staining (d, epifluorescence) co-localise.
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MAX2 is required at each node to suppress axillary bud growth
Root–shoot grafting demonstrates that MAX2 is required in the shoot to repress branching (Booker et al., 2005). To determine the site(s) of action of MAX2 in the shoot, we produced chimeric shoots carrying max2 mutant sectors in a phenotypically wild-type (MAX2/max2) background and studied their axillary bud growth.
As a tool to generate easily detectable, colour-marked sectors, we used the Arabidopsis cell autonomy (CAUT) lines. They employ the CHLORATA-42 gene (CH-42), normally located on chromosome IV, as a marker. chlorata-42 is a recessive, homozygous viable T-DNA insertion allele (Koncz et al., 1990). The yellow ch-42 mutant phenotype manifests in all cells that produce chloroplasts and is cell autonomous (Furner, 1996; Furner et al., 1996). The CAUT lines were generated by transformation of ch-42 homozygous plants with the dominant (green) CH-42 wild-type allele. The individual transformant lines were extensively backcrossed to the yellow (ch-42) parent until they segregated 1:1. Such single-insert lines were made homozygous and the inserts mapped using recombinant inbred lines (Lister and Dean, 1993), gridded bacterial artificial chromosomes (BACs; Choi et al., 1995; Liu et al., 1995, 1999; Mozo et al., 1998) or flanking DNA sequence. Seventy-six lines are available, each with a correcting insert at a single mapped site. In effect this means that the capacity to produce chlorophyll, and hence a cell-autonomous colour marker, is translocated to a different site in each line. To use the CAUT lines to study cell autonomy, the mutant of interest is backcrossed into the homozygous yellow ch-42 mutant background and then to a CAUT line with a correcting insert near the wild-type copy of the locus. X-irradiation of F1 or F2 seeds results in yellow sectors on the plant derived from the L2 and/or L3 layers of the seed SAM. Such sectors have usually lost both the correcting insert and the adjacent wild-type copy of the gene of interest and are sectorial chimeras with marked mutant tissue surrounded by wild-type tissue and overlain by a wild-type (L1-derived) colourless epidermis.
To study the cell autonomy of the MAX2 gene, we generated the ch-42 max2 double mutant. This was crossed with either of two CAUT lines carrying CH-42 inserted close to the MAX2 locus on chromosome II (7F and A24, see Experimental procedures). The resulting F1 (and half of the F2, which is more easily bulked up) has the target genotype for sector generation shown in Figure 3(a). These plants show repressed, wild-type branching and are green. Irradiation-induced deletion of the linked wild-type copies of MAX2 and CH-42 from one chromosome II homologue (red in Figure 3a) in a meristematic cell will result in yellow-marked, max2 somatic sectors. We irradiated the dry F2 seed to induce chromosomal deletions in embryonic SAM cells. Sectors on the first leaf pair are typically frequent but very small, sectors affecting later leaves are typically larger but less frequent and sectors affecting late leaves are typically very large and infrequent (Furner and Pumfrey, 1992; Irish and Sussex, 1992). This is because the cells at the periphery of the dry seed SAM give rise to the very early leaves and later leaves are derived from cells that undergo proliferation in the meristem before leaf initiation. Cells at the centre of the dry seed SAM can take over the meristem over time and the whole layer of the meristem will become of a single marked genotype late in development (Furner, 1996).
Figure 3. MAX2 is required at each node to repress bud growth. (a) Genotype of the irradiated plants. Note the plant is mutant (ch-42) at the authentic locus on chromosome IV and the green phenotype is conferred by the transgene CH-42 insert on chromosome II. Radiation-induced loss of the transgene also results in loss of the wild-type copy of MAX2 (red region) resulting in yellow ch-42 tissue hemizygous for the max2 mutation. Sectors can occur in any tissue but will only be seen if they affect chlorophyll-producing tissue. (b, c) Sector-free rosettes with a wild-type phenotype. Arrows in (c) indicate the small axillary buds. (d) Rosette with a large yellow max2 sector on the left. The apex of the plant and the youngest leaves were removed. Axillary buds in the sector have produced leaves about half the length of the rosette leaves (arrowheads). No axillary leaves are visible in the wild-type part of the rosette. (e) Rosette in which one of the oldest leaves carried a yellow max2 sector which extended into the axil. The yellow leaves (arrowheads) were produced by the bud that developed in this axil. (f) Rosette in which several parastichious leaves carried a narrow yellow max2 sector which extended into the axil. The photograph shows one of the sectored rosette leaves (white arrow) with its axillary bud. The bud centre and most axillary leaves (white arrowheads) are yellow, but some of the outer axillary leaves are chimeric or green (black arrowheads).
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Wild-type and max2 vegetative rosettes show a pronounced difference in axillary bud size over a wide range of node positions along the shoot axis (Stirnberg et al., 2002). Furthermore, large vegetative sectors may be generated from cells at the centre of the embryonic SAM when flowering is delayed (Furner et al., 1996). Therefore, the irradiated F2 was grown in short photoperiods to prolong vegetative growth of the plants. F2 individuals with large yellow ch-42 sectors affecting the (L2-derived) leaf tissue and non-sectored F2 controls (green MAX2 and yellow max2 individuals) were selected and transferred to individual pots for further growth and observation.
Figure 3(b) shows the vegetative rosette of a green MAX2 control 9 weeks after sowing, when phenotypic analysis was carried out. The axillary buds were about 1 mm in length, lacking expanding axillary leaves (Figure 3c). Development of yellow ch-42 max2 control plants was significantly delayed due to chlorophyll deficiency, but the enlarged axillary buds with expanding axillary leaves typical of max2 appeared later. No delay in the development of ch-42 max2 tissue was observed in chimeric plants, where extensive ch-42 max2 sectors were maintained by wild-type tissue (Figure 3d). Table 1 lists the chimeric F2 plants (A–N) for which the phenotypic analysis was performed and for which the max2 mutant sector genotype was confirmed by genetic analysis of sector F3 progeny. In individuals A–E, the sector consisted of many rosette leaves and either the whole SAM (A–C) or part of the SAM (D, E, Figure 3d), which continued to produce yellow max2 leaves. In individuals F–H, several yellow max2 leaves had been produced, but the sector tissue had become eliminated from the SAM, which was only producing green, wild-type leaves at the time of analysis. Individuals I–N carried more narrow sectors which affected the central part of one (K–N, Figure 3e) or several leaves (I, J, Figure 3f). In most individuals, the mutant sector included both the L2 (sub-epidermal) and L3 (central) tissue layer except for C, E and F, where sector leaves with a pale green centre and yellow margins indicated that only the L2 was ch-42 max2. In Arabidopsis, the green tissue of the axillary bud originates from two or more L2 cells at the base of the subtending leaf (Furner and Pumfrey, 1992; Irish and Sussex, 1992). Therefore, a leaf completely included in a mutant sector should carry a mutant bud. If only part of the leaf carries a sector, a central sector is likely to extend into the petiole and include the axillary bud (Furner and Pumfrey, 1992). There are no data on the colourless (L1 derived) epidermis in these situations, but it can reasonably be presumed that these mutant buds have a wild-type epidermis. The recovery of non-L2 gametes (see below) from some plants implies that this is the case.
Table 1. Genetic mosaic analysis of MAX2 action in the shoot. List of chimeric shoots bearing max2 sectors
|F2 individuala||Shoot organs included in sector||Leaf tissue layers contributing to sector||Sector bud colourb||Sector bud size||Non-sector bud size||Phenotypic segregation in F3 from a bud in sectorb|
|Leaves||Shoot apex||g MAX2||y max2||Others|
|A||Many||Whole||L2 and L3||y||Large||Small||1||25||1|
|B||Many||Whole||L2 and L3||y||Large||Small||0||22||0|
|D||Many||Part||L2 and L3||y||Large||Small||0||24||0|
|G||Several||–||L2 and L3||y/chimeric||Large||Small||0||26||0|
|H||Several||–||L2 and L3||y||Large||Small||12||26||0|
|I||Parts of several||–||L2 and L3||Chimeric||Large||Small||0||24||0|
|J||Parts of several||–||L2 and L3||Chimeric||Large||Small||0||28||0|
|K||Part of one||–||L2 and L3||y||Large||Small||0||19||0|
|L||Part of one||–||L2 and L3||Chimeric||Large||Small||0||28||0|
|M||Part of one||–||L2 and L3||y||Large||Small||1||26||0|
|N||Part of one||–||L2 and L3||y||Large||Small||0||24||0|
In all sectored plants, independent of sector size, axillary buds in the max2 sector were large, with several expanding leaves. Axillary buds outside the sector were wild-type (Table 1, Figure 3d–f). Thus sector bud phenotype corresponded to sector genotype, and was not affected by wild-type MAX2 function in the epidermis or other shoot or root tissue. Sector axillary buds subtended by a completely mutant leaf (Figure 3d) were indistinguishable from those subtended by a leaf with part of the lamina green (Figure 3f). Thus, MAX2 activity in the lamina is not sufficient to rescue the max2 bud phenotype. Rather, MAX2 may act in or close to the axillary bud to repress its growth. Some of the non-repressed axillary buds were chimeric, i.e. contained some green MAX2 tissue (Table 1 and Figure 3f). This occurred when the leaf subtending the bud included a sector boundary, and likely mutant and non-mutant cells participated in formation of these axillary buds. These buds had green or chimeric outer axillary leaves, but the younger axillary leaves were yellow (Figure 3f). This further delimits the tissue requiring MAX2 for bud repression to the central tissues of the bud, or tissue of the leaf or primary shoot axis close to the bud, and hints that max2 tissue may exert non-autonomous effects on adjacent wild-type tissue, incorporating adjacent non-mutant tissue into the axillary shoot.
The loss of the wild-type MAX2 copy in the yellow-marked sector tissue was confirmed after phenotypic analysis. Shoots were decapitated above the sector and shifted to long photoperiods to encourage outgrowth of inflorescences from the sector. Loss of the linked CH-42 and MAX2 wild-type copies should result in uniformly yellow max2 F3 sector progeny. This was confirmed for the majority of the 14 chimeric plants (Table 1). However, green wild-type MAX2 segregants were found in F3 sector progeny from three chimeric plants (A, H, M), the proportion being significantly lower than the 75% expected had deletion of MAX2 and CH-42 not occurred. These segregants may have resulted from invasion of the sector L2 lineage by wild-type cells from the epidermal L1 layer during formation of the axillary branch from which the F3 seed was collected. Such non-L2 gametes were also found in earlier studies of cell autonomy (Bouhidel and Irish, 1996; Furner et al., 1996). For a few F3 segregants, branching or colour phenotype could not be classified (individuals A, C). These might have carried additional radiation-induced mutations severely affecting development.
In conclusion, the sector analysis shows that MAX2 is required in the green (L2-derived) tissue at each individual node for repression of its associated axillary bud, and that it acts either in the bud itself or close to it.
MAX2 overexpression partially rescues the branching phenotype of max1, max3 and max4
To investigate whether the level of MAX2 in the plant is limiting for repression of branching, we expressed MAX2 in plants under control of the strong CaMV 35S promoter (35S::M2). First, we tested whether the overexpression construct complements the max2 mutant phenotype. Thirty-nine of 42 35S::M2 (max2) primary transformants had a wild-type phenotype. From these, 10 independent lines containing a single insert were brought to homozygosity. For nine lines, branching was reduced to wild-type level and RT-PCR analysis showed weak to moderate increases of MAX2 transcript level compared with untransformed max2. One line was not rescued, but the endogenous plus transgenic MAX2 transcript level was not increased in comparison with untransformed max2 (see Figure S1a). Thus, the 35S::M2 construct is functional and directs MAX2 expression in those tissues where it is required to repress branching.
For 35S::M2 in the wild-type background, we obtained 30 independent primary transformants. None of these were noticeably different from the wild type. From these, 11 lines were taken to homozygosity. Two homozygous lines showed a significant increase in MAX2 transcript level, but branching did not differ significantly from wild type in long photoperiods (data not shown). The highest-expressing line was further compared with the wild type in a decapitation assay designed to detect small alterations in shoot branching (Figure 4). Plants were grown in short photoperiods for 30 days to increase the number of vegetative nodes, outgrowth from which was encouraged by decapitation of the primary inflorescence after induction of flowering in long photoperiods. Again, branching from the rosette was not significantly different between wild type and the high-expressing 35S::M2 (wt) line (Figure 4a). In leaf samples taken at the end of the experiment, the detection threshold of MAX2 transcript by RT-PCR was reached at least five cycles earlier in this 35S::M2 line than in wild type, indicating a significant rise in transcript level (Figure 4b). This suggests that the level of MAX2 is not limiting for repression of branching in wild-type plants.
Figure 4. The effect of overexpressing MAX2 in wild-type (wt), max1, max3 and max4 genetic backgrounds. (a) Branching of wild type, max1, max3 and max4 in the absence (untransformed) or in the presence of MAX2 overexpression from the 35S promoter (35S::M2). Branching was assessed in a decapitation assay (see Experimental procedures). The mean number of rosette branches with a length of at least 2 cm 10 days after decapitation is shown (error bar = SEM, n = 11). (b) Analysis of MAX2 expression for the experiment presented in (a). Reverse transcriptase-PCR from total RNA of rosette leaves, collected after branching had been assessed, using primers that amplify endogenous plus transgenic MAX2. Detection of ACTIN2 transcript was used as a cDNA normalization control. cyc, cycle number.
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Grafting and double mutant analysis suggest that the MAX genes define a signalling pathway that results in repression of bud growth, in which MAX2 acts in the downstream response to the signal synthesized by the action of MAX1, MAX3 and MAX4 (Booker et al., 2005). To test this hypothesis further, we asked whether MAX2 overexpression suppresses the branching phenotype of max1, max3 or max4. The highest-expressing 35S::M2 (wt) line (selective marker kanamycin resistance) was crossed with these mutants and with max2. Forty kanamycin-resistant F2 individuals were selected for each cross. Amongst these, we could identify homozygous max mutants, bushier than wild type, for the crosses with max1, max3 and max4. The proportions were not significantly different from 0.25. F3 seed was collected from these max mutant F2 individuals and a family homozygous for the 35S::M2 insert selected. For the cross with max2, all kanamycin-resistant F2 plants were wild type. As MAX2 and MAX3 are closely linked (Booker et al., 2004; Stirnberg et al., 2002), max2 and max3 should recombine with 35S::M2 with similar frequencies. Therefore, the lack of max2 segregants must be due to mutant rescue, confirming that the 35S::M2 transgene used in the crosses was functional. Shoot branching of max1, max3 and max4 in the presence and absence of 35S::M2 was compared in the decapitation assay described above (Figure 4a). 35S::M2 partially rescued the increased branching of max1, max3 and max4. Using leaf samples collected at the end of the assay, we confirmed that MAX2 overexpression in the max, 35S::M2 double homozygotes was at a level similar to the 35S::M2(wt) line (Figure 4b).
This result suggests that the level of MAX2 becomes limiting for repression of branching when the inhibitory signal produced by MAX1, MAX3 and MAX4 is low, and is consistent with an action of MAX2 downstream of MAX1, MAX3 and MAX4. The incomplete rescue of max1, max3 and max4, in spite of a substantial rise in MAX2 transcript level, indicates that the signal produced by MAX1, MAX3 and MAX4 acts to promote MAX2 action in some way other than transcriptional upregulation.
F-box-deleted MAX2 has a dominant-negative effect on shoot branching
SCF-type E3 ubiquitin ligases are multiprotein complexes, in which core subunits with catalytic and scaffold function (ubiquitin-conjugating enzyme E2, Skp1, Cullin, Rbx1) combine with a variable F-box protein subunit that confers the substrate specificity (Cardozo and Pagano, 2004; Willems et al., 2004). Two functional domains enable F-box proteins to mediate substrate ubiquitination. One domain binds the substrate protein. In MAX2, the C-terminal leucine-rich repeat region probably fulfils this role. The second domain, the F-box, is required for assembly of the SCF complex by binding to Skp1. An F-box-homologous region is located at the N-terminus of MAX2 (Stirnberg et al., 2002; Woo et al., 2001).
To test for the significance of the F-box domain for MAX2 protein function, we constructed 35S::ΔF–M2, and introduced it into max2 plants. This construct was identical to 35S::M2, except that the codons for amino acids 9–47 of the protein, spanning the F-box, were deleted. All 15 35S::ΔF–M2 (max2) primary transformants had a mutant phenotype. Seven independent, single-insert lines were brought to homozygosity. Despite weak to moderate increases in endogenous plus transgenic MAX2 transcript, their branching was not reduced compared with untransformed max2 (Figure S1b). This suggests that deletion of the F-box abolishes the function of the MAX2 protein.
Deletion of the F-box domain may leave substrate protein binding activity unaffected. It has been reported that co-expression of such ΔF deletion versions with the endogenous F-box-containing protein interferes with ubiquitination of the target protein, resulting in its stabilization and increased activity (Hart et al., 1999; Kitagawa et al., 1999; Marikawa and Elinson, 1998; Wu et al., 2001; Yaron et al., 1998). To test for such dominant-negative action, 35S::ΔF–MAX2 was transformed into wild-type plants. We obtained 28 primary transformants, which were either indistinguishable from wild type, or showed somewhat increased branching. Seven single-insert lines, including both phenotypic groups, were made homozygous. The analysis of their branching, and their endogenous and transgenic (ΔF) MAX2 transcript levels, in comparison with wild type and max2, is shown in Figure 5. Three lines (98, 101, 106) showed a significant increase in branching (about twice the number of branches compared to wild type) and had an intermediate level of ΔF–MAX2 transcript. Four lines had a branching pattern indistinguishable from the wild type. The two lowest (100, 102), but also the two highest, ΔF–MAX2-expressing lines (103, 104) belonged to this group. Similar results were obtained in two further independent repeats of this experiment (data not shown). The highest-expressing lines (103, 104) showed no mutant rescue when crossed into the max2 mutant background, ruling out the possibility that the F-box becomes dispensable for MAX2 function at this level of expression (data not shown).
Figure 5. Expression of F-box-deleted MAX2 in the wild-type (wt) background. (a) Branching in wild type, max2 and seven independent homozygous lines expressing F-box-deleted MAX2 from the 35S promoter (35S::ΔF–M2). Branching was assessed as in Figure 4(a), n = 9–10. (b) Analysis of endogenous MAX2 and transgenic, F-box-deleted MAX2 expression for the experiment presented in (a). Reverse transcriptase-PCR from total RNA of rosette leaves, collected after branching had been assessed. Reverse priming in their divergent terminators was used to amplify selectively endogenous and transgenic MAX2 transcripts. Detection of ACTIN2 transcript was used as a cDNA normalization control. cyc, cycle number.
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Apart from a dominant-negative action at the protein level, introduction of the 35S::ΔF–M2 transgene could have caused an identical phenotypic effect by co-suppressing the endogenous, functional MAX2 copy. None of the 35S::ΔF–M2 lines showed a substantial reduction of endogenous MAX2 transcript compared to untransformed wild type (Figure 5b). Endogenous MAX2 expression tended to be highest in the two highest-expressing 35S::ΔF–M2 lines, 103 and 104. It tended to be lowest in the three 35S::ΔF–M2 lines that showed increased branching, but was not lower than in wild-type plants in two experiments and only slightly lower in the third.
MAX2 interacts with core SCF subunits in vivo
Two experimental approaches have been taken to examine whether ORE9/MAX2 participates in an SCF complex. In a yeast two-hybrid assay, the F-box domain of ORE9 interacted with the Skp1-like Arabidopsis protein ASK1; furthermore, in vitro translated ORE9 bound purified GST-ASK1 fusion protein in an F-box-dependent manner (Woo et al., 2001). We investigated whether MAX2 interacts with core SCF subunits in planta. A C-terminally myc epitope-tagged version of MAX2, under control of the 35S promoter, was expressed in the max2 mutant (35S::M2-myc). In contrast to 35S::M2 primary transformants, few 35S::M2-myc transformants showed complete rescue of the branching phenotype. Analysis of MAX2 transcript levels indicated that the highest-expressing 35S::M2-myc lines showed the best rescue (data not shown), suggesting that the epitope tag reduces protein function but does not abolish it completely. A high-expressing, completely rescued 35S::M2-myc line was used for co-immunoprecipitation analysis (Figure 6). The SCF subunits ASK1 and AtCUL1 (an Arabidopsis Cullin) were detected in protein extracts from non-transgenic max2 and from max2 expressing MAX2-myc. There were no differences in expression levels of these proteins between both lines. Co-immunoprecipitation of ASK1 and AtCUL1 with anti-c-myc antibody depended on the presence of MAX2-myc in the extract. Cycles of covalent attachment of the small protein RUB1 to AtCUL1, followed by its removal, are crucial for optimal SCF ubiquitination activity (Parry and Estelle, 2004). The two bands detected with anti-CUL1 antiserum are therefore probably RUB1-modified and unmodified AtCUL1, which both co-immunoprecipitated with MAX2-myc. The ASK1 polyclonal antiserum we used cross-reacts with ASK2, which is a protein closely homologous and functionally redundant to ASK1 (Gagne et al., 2002; Liu et al., 2004). MAX2-myc appeared to interact with both Skp1-like proteins. This experiment demonstrates the interaction of MAX2 with core SCF components in vivo.
Figure 6. MAX2 interacts with SCF core components ASK1 and AtCUL1 in vivo. Anti-myc immunoprecipitates were prepared from total protein extracts of max2 and a transgenic line expressing myc-tagged MAX2 in the max2 background [35S::M2-myc (max2)]. Total protein extracts (lanes 1, 2) and immunoprecipitates (lanes 3, 4) were analysed by Western blotting and probing with polyclonal antiserum raised against AtCUL1 (top) and against ASK1 (bottom).
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