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. 2004 Jun;16(6):1550–1563. doi: 10.1105/tpc.019224

SPINDLY and GIGANTEA Interact and Act in Arabidopsis thaliana Pathways Involved in Light Responses, Flowering, and Rhythms in Cotyledon Movements

Tong-Seung Tseng a,1, Patrice A Salomé b, C Robertson McClung b, Neil E Olszewski a,2
PMCID: PMC490045  PMID: 15155885

Abstract

SPINDLY (SPY) is a negative regulator of gibberellin signaling in Arabidopsis thaliana that also functions in previously undefined pathways. The N terminus of SPY contains a protein–protein interaction domain consisting of 10 tetratricopeptide repeats (TPRs). GIGANTEA (GI) was recovered from a yeast two-hybrid screen for proteins that interact with the TPR domain. GI and SPY also interacted in Escherichia coli and in vitro pull-down assays. The phenotypes of spy and spy-4 gi-2 plants support the hypothesis that SPY functions with GI in pathways controlling flowering, circadian cotyledon movements, and hypocotyl elongation. GI acts in the long-day flowering pathway upstream of CONSTANS (CO) and FLOWERING LOCUS T (FT). Loss of GI function causes late flowering and reduces CO and FT RNA levels. Consistent with SPY functioning in the long-day flowering pathway upstream of CO, spy-4 partially suppressed the reduced abundance of CO and FT RNA and the late flowering of gi-2 plants. Like gi, spy affects the free-running period of cotyledon movements. The free-running period was lengthened in spy-4 mutants and shortened in plants that overexpress SPY under the control of the 35S promoter of Cauliflower mosaic virus. When grown under red light, gi-2 plants have a long hypocotyl. This hypocotyl phenotype was suppressed in spy-4 gi-2 double mutants. Additionally, dark-grown and far-red-light–grown spy-4 seedlings were found to have short and long hypocotyls, respectively. The different hypocotyl length phenotypes of spy-4 seedlings grown under different light conditions are consistent with SPY acting in the GA pathway to inhibit hypocotyl elongation and also acting as a light-regulated promoter of elongation.

INTRODUCTION

SPINDLY (SPY) was identified in genetic screens for negative regulators of gibberellin (GA) signaling in Arabidopsis thaliana (Jacobsen and Olszewski, 1993). Recessive spy mutations suppress all of the phenotypes caused by GA deficiency (Jacobsen and Olszewski, 1993; Wilson and Somerville, 1995; Jacobsen et al., 1996; Swain et al., 2001). Because spy plants exhibit phenotypes, including reduced hypocotyl length of dark-grown seedlings, that are not readily attributable to defects in GA responses, it has been suggested that SPY has additional undefined function(s) (Swain et al., 2001).

SPY has significant similarity to animal O-linked β-N-acetylglucosamine transferase (OGT) (Thornton et al., 1999b; Roos and Hanover, 2000), an enzyme that catalyzes posttranslational addition of a single O-linked β-N-acetylglucosamine to specific Ser/Thr of cytosolic and nuclear proteins (Wells et al., 2001). Consistent with SPY being an OGT, it has OGT activity, and spy mutants have changes in protein GlcNAcylation (Thornton et al., 1999a).

There is much evidence from animals indicating that O-GlcNAcylation of proteins is a regulatory modification (Hart, 1997; Comer and Hart, 2000; Wells et al., 2001). The modification is dynamic and the extent of modification is affected by metabolic and hormonal signals. The modification affects the half-life and localization of proteins. It has been shown for several proteins that specific Ser and/or Thr residues can be alternatively phosphorylated or GlcNAcylated, suggesting that one way GlcNAcylation regulates protein activity is by affecting the phosphorylation status.

Protein GlcNAcylation is essential in both plants and animals. Deletion of the OGT gene of mouse causes lethality (Shafi et al., 2000). Double mutants affected in both SPY and SECRET AGENT, the other Arabidopsis OGT, die during embryogenesis (Hartweck et al., 2002). Loss of SPY and SECRET AGENT activity also reduces the viability of the female and male gametes.

Both SPY and OGT have two distinct domains, the C-terminal catalytic domain and the N-terminal tetratricopeptide repeats (TPRs) (Jacobsen et al., 1996; Roos and Hanover, 2000; Olszewski et al., 2002). TPRs are degenerate 34–amino acid motifs that act as scaffolds for assembly of multiprotein complexes (Blatch and Lassle, 1999). Deletion of the TPRs converts OGT from a homotrimer to a monomer and affects substrate specificity (Kreppel and Hart, 1999; Lubas and Hanover, 2000). Several spy alleles have missense or small in-frame deletions in the TPR domain (Jacobsen et al., 1996; Tseng et al., 2001), and ectopic overexpression of the TPR domain produces spy-like phenotypes (Tseng et al., 2001), suggesting that protein interactions at this domain are critical to the functioning of SPY.

To further investigate the role of the TPR domain of SPY, we conducted yeast two-hybrid screening to identify proteins that interact with SPY. One protein identified in this screen was GIGANTEA (GI).

GI is a novel nuclear-localized protein (Huq et al., 2000) that is involved in several processes (Araki and Komeda, 1993; Koornneef et al., 1998; Fowler et al., 1999; Park et al., 1999; Huq et al., 2000), including the induction of flowering by long days, inhibition of hypocotyl elongation by red light, and the circadian clock. When grown under long days, gi mutants flower late. CONSTANS (CO) and FLOWERING LOCUS T (FT) function downstream of GI. CO and FT RNA are less abundant in gi mutants, and overexpression of CO suppresses the late flowering of gi plants (Suárez-Lopez et al., 2001). Because gi mutants have a long hypocotyl when growing under red light (Huq et al., 2000), GI is thought to function in the phytochrome B signaling pathway. Circadian rhythms in gene expression, leaf movement, and transpiration are affected in gi mutants. Some gi alleles decrease the free-running period, whereas other alleles increase this period (Park et al., 1999). The amplitudes of rhythms rapidly damps out when gi plants are removed from entrainment (Park et al., 1999; Sothern et al., 2002).

The hypothesis that GI and SPY function as a complex was tested by examining the flowering time, pattern of CO and FT expression, and responses to red light of spy-4 gi-2 double mutants. We also examined the rhythm in cotyledon movement of several spy mutants and a spy-4 gi-2 double mutant. The results from these experiments were consistent with the hypothesis that SPY and GI function together.

RESULTS

The SPY TPR Domain Interacts with GI

The N terminus of SPY contains 10 TPR repeats. TPRs mediate interactions with other proteins (Blatch and Lassle, 1999). Previously, the TPRs of SPY were shown to participate in SPY–SPY interactions (Tseng et al., 2001). Because the large number of SPY TPRs suggested that SPY could interact with additional proteins, we conducted a yeast two-hybrid screen for Arabidopsis proteins that interact with this domain. A clone encoding the C-terminal third (amino acids 788 to 1173) of GI was recovered multiple times. Although the interaction between GI and the TPR domain of SPY was sufficient to activate LacZ and HIS3, the two reporter genes used in this assay, no activation of these genes occurred when GI was tested with the TPR domain of Mbb1 (Vaistij et al., 2000), an unrelated protein from Chlamydomonas reinhardtii (Figure 1A), suggesting that the interaction is specific to the TPR domain of SPY. The interaction also occurred when GI and SPY were reversed as bait and prey and when full-length SPY was used as a bait (Figure 1B).

Figure 1.

Figure 1.

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The TPR Domain of SPY Interacts with GI.

(A) GI interacts with SPY but not with Mbb1. Yeast cells were grown on selecting medium lacking histidine (−HIS) to test the auxotrophy. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside was added to test for the expression of β-galactosidase reporter gene. AD- and AD-GI indicate cells expressing the GAL4 activation domain alone or the GAL4 activation domain fused to the C terminus of GI. TPR, Mbb1, SPY, and DB- indicate cells expressing the TPR domain of SPY, the TPR domain of Mbb1 or SPY as a fusion with the GAL4 DNA binding domain, or the DNA binding domain alone.

(B) 2-Nitrophenyl β-d-galactopyranoside hydrolysis assays of β-galactosidase (β-Gal) reporter gene expression show that the interaction between SPY and GI occurs when SPY is the bait or prey. The minus sign indicates empty vectors used as negative controls.

(C) The β-galactosidase (β-Gal) activity induced by interactions involving the wild-type TPR domain and spy-2 TPR domain. Data from one of three independent experiments are shown. Similar trends of β-galactosidase activity were observed in all three experiments. The standard deviation is indicated on each bar; however, in some cases it is too small to be visible.

The TPR domain of SPY interacted with GI in an in vitro pull-down assay (Figure 2A). In three independent in vitro interaction assays, more 35S-labeled GI protein interacted with a maltose binding protein (MBP)-SPY TPR fusion than with MBP alone. In addition, full-length SPY and GI were found to interact in Escherichia coli cells (Figure 2B). MBP and MBP-GI were coexpressed in E. coli with S-tagged full-length SPY. When MBP and MBP-GI were affinity purified from cell-free lysates, S-tagged SPY copurified with MBP-GI but not MBP. Similar results were obtained in three independent experiments. Full-length SPY also interacted with GI in an in vitro pull-down assay (data not shown).

Figure 2.

Figure 2.

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GI Interacts with SPY in Pull-Down Assays.

(A) Lanes 1 and 2 of the top panel show the 35S-labeled GI that interacted with the MBP or the MBP-TPR fusion protein (Tseng et al., 2001), respectively. Ten percent of the supernatant from the interaction reaction was loaded onto lane 3. The bottom panel shows a Coomassie blue–stained gel containing the amounts of the MBP and MBP-TPR (TPR) proteins used in the interaction assay. Note that MBP-TPR pulled down more GI than MBP even though more MBP was present in the assay.

(B) MBP or MBP-GI were affinity purified from E. coli coexpressing S-tagged SPY. The affinity-purified proteins and any copurified proteins were resolved by SDS-PAGE and blotted. In the top panel, S-tagged SPY was detected using anti-S antibodies. S-SPY indicates full-length S-tagged SPY, and the bracket indicates breakdown products. Lanes 1 and 2 contain MBP and MBP-GI and proteins that affinity purified with them, respectively. Lanes 3 and 4 contain the whole-cell lysates that were used for the affinity purification in lanes 1 and 2, respectively. In the bottom panel, the membrane from the top panel was stripped and probed with anti-MBP antibodies, which revealed that similar amounts of MBP and MBP-GI were present in the samples.

The experiments in (A) and (B) were repeated three times with similar results.

The spy-2 Mutation Differentially Affects SPY-SPY versus SPY-GI Interactions

The spy-2 mutation causes missplicing of SPY RNA, leading to deletion of portions of TPRs 8 and 9 (Jacobsen et al., 1996). The effects of the spy-2 mutation on the interaction of SPY with wild-type SPY TPR and GI were examined (Figure 1C). The spy-2 mutation weakened the interaction with wild-type SPY TPR and increased the interaction with GI.

SPY Acts with GI in Regulating Hypocotyl Elongation

GI encodes a novel nuclear protein that participates in responses, including photoperiodic induction of flowering, functioning of the circadian clock, and inhibition of hypocotyl elongation by red light (Araki and Komeda, 1993; Koornneef et al., 1998; Fowler et al., 1999; Park et al., 1999; Huq et al., 2000). gi seedlings are hyposensitive to red light and exhibit a long hypocotyl phenotype when grown under red light (Huq et al., 2000).

Evidence that the interaction between SPY and GI plays a functional role was sought by determining the length of the hypocotyl of wild-type, spy-4, gi-2, and spy-4 gi-2 double mutants growing under different light conditions. Under red light, gi-2 exhibited the expected long hypocotyl phenotype, whereas spy-4 and the double mutant were indistinguishable from the wild type (Figure 3). This result is consistent with spy-4 being epistatic to gi-2 and, furthermore, suggests that GI and SPY function in a common pathway.

Figure 3.

Figure 3.

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The Effect of spy-4 and gi-2 on Hypocotyl Elongation.

Hypocotyl length, mean ± se (n ≥ 15), of 6-d-old seedlings grown in continuous far-red light (FR), continuous red light (R), or in the dark (D).

The hypocotyl of dark-grown gi-2 was indistinguishable from the wild type but was slightly longer than the wild type when grown under far-red light (Figure 3). This long hypocotyl phenotype of gi-2 has been seen previously (see Figure 1B in Huq et al., 2000). As expected (Swain et al., 2001), spy-4 seedlings exhibited a short hypocotyl phenotype in the dark, but surprisingly, plants grown under far-red illumination had a long hypocotyl. The hypocotyl of spy-4 gi-2 plants grown under dark or far-red illumination was indistinguishable or slightly longer than that of spy-4.

These results suggest that SPY has several roles in hypocotyl elongation (Figure 4A). One of these roles is to negatively regulate GA signaling and the second is to act as a red-light-inhibited promoter of elongation. Because far-red light inhibits GA biosynthesis (Reid et al., 2002), we hypothesized that the long hypocotyl phenotype of far red–grown spy-4 seedlings was because of the loss of inhibition of GA signaling. This hypothesis requires that the loss of SPY's negative regulation of GA signaling has a greater impact on hypocotyl length than the loss of the positive role. A prediction of this hypothesis is that negating the far-red-light–induced reduction in GA levels by supplying GA3 to wild-type and spy-4 seedlings will reveal the positive role of SPY in hypocotyl elongation. This prediction was tested by examining the hypocotyl length of wild-type, gi-2, spy-4, and spy-4 gi-2 seedlings grown on different concentrations of GA3 (Figure 5). As predicted by the model, at the highest concentration of GA3, spy-4 and spy-4 gi-2 hypocotyls were shorter than the wild type and gi-2.

Figure 4.

Figure 4.

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Role of the SPY-GI–Interacting Complex in Regulating Hypocotyl Elongation and Flowering Time.

(A) SPY negatively regulates GA-promoted hypocotyl elongation and also acts independently of GA to promote hypocotyl elongation. Red light illumination acting through phytochrome B (phyB) activates GI, which in turn inactivates SPY that is complexed with it, leading to a reduction in hypocotyl elongation. Far-red light inhibits GA biosynthesis, thereby reducing hypocotyl elongation. In the dark, GA responses are saturated (Gendreau et al., 1999; Swain et al., 2001). Under both far-red light and in the dark, the positive role of SPY in hypocotyl elongation is active because SPY is not inhibited by GI.

(B) SPY acts as a negative regulator in both the long-day and GA pathways to regulate flowering time. Interaction of GI with SPY inhibits SPY, allowing flowering to occur.

Figure 5.

Figure 5.

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Treatment with GA3 Reveals a Positive Role for SPY in Hypocotyl Elongation of Seedlings Grown under Continuous Far-Red Light.

The hypocotyl length of the wild type (square) and spy-4 (circle), gi-2 (diamond), and spy-4 gi-2 (triangle), mean ± se (n ≥ 27), of 6-d-old seedlings grown on medium supplemented with different amounts of GA3 under continuous far-red light.

spy Suppresses the Late Flowering and Reduced CO and FT RNA Levels of gi

Flowering in Arabidopsis can be induced by environmental signals, including photoperiod, and endogenous pathways, including the GA-signaling pathway (Mouradov et al., 2002; Simpson and Dean, 2002). GI functions in the long-day pathway (Araki and Komeda, 1993; Koornneef et al., 1998; Fowler et al., 1999). The flowering time of gi plants grown under inductive long-day conditions is delayed. spy plants flower early. Although activation of the GA pathway in spy plants is likely, at least in part, to account for the early flowering of these mutants (Jacobsen and Olszewski, 1993; Jacobsen et al., 1996; Swain et al., 2001), the interaction between SPY and GI raises the possibility that SPY also acts in the long-day pathway. To determine if SPY acts in the long-day pathway, the effects of daily treatment with high doses of GA3 and spy-4 on the flowering time of the wild type and gi-2 in the Columbia background and gi-3 in the Landsberg erecta (Ler) background were compared (Figure 6). In this experiment, plants were sprayed daily to runoff with 5 × 10−4 M GA3, a treatment regime that suppresses the delayed flowering of ga1, an extreme GA biosynthesis mutant (Sun and Kamiya, 1994), more effectively than spy-4 (Figure 7). Consistent with SPY acting in both the GA signaling and long-day pathways, spy-4 more effectively suppressed the late flowering of gi-2 and gi-3 than treatment with GA3.

Figure 6.

Figure 6.

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The Late Flowering Phenotype of gi Is Suppressed More Effectively by spy Than by Treatment with GA3.

(A) and (C) Flowering time (total leaf number) of Arabidopsis plants in the Columbia (Col) (A) and Ler backgrounds (C). Closed and hatched bars indicate the number of rosette leaves, and open bars indicate the number of cauline leaves. Plants grown under long days were treated daily with either 5 × 10−4 M GA3 (hatched/open bars) or with 1.6% ethanol (closed/open bars). Data represent the mean ± sd (n ≥ 10).

(B) and (D) The data from (A) and (C), respectively, are expressed as percentage of suppression to the wild type. For untreated plants, percentage of suppression to the wild type = (total leaf number of gi – total leaf number of the double mutant)/(total leaf number of gi – total leaf number of the wild type) × 100. For GA3 treated plants, percentage of suppression to the wild type = (total leaf number of gi – total leaf number of GA3-treated gi)/(total leaf number of gi – total leaf number of GA3-treated wild type) × 100.

Figure 7.

Figure 7.

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Treatment with 5 × 10−4 M GA3 More Effectively Suppresses the Late Flowering of ga1 Than spy-4.

Wild-type, ga1-2, spy-4, and spy-4 ga1-2 plants, all in the Columbia (Col) background (Swain et al., 2001), were grown under long-day conditions (16L/8D). One set of ga1-2 plants was treated daily with 5 × 10−4 M GA3 (hatched/open bars). Another set of ga1-2 plants and the other plants were treated with 1.6% ethanol (closed/open bars). Data represent the mean ± sd (n ≥ 8). The ga1-2 plants that were sprayed with 1.6% ethanol had not flowered when the experiment was terminated.

CO and FT are positive-acting components of the long-day pathway (Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000; Suárez-Lopez et al., 2001). CO acts downstream of GI and upstream of FT. The effects of spy-4 and GA3 treatment on the flowering time of co-2 and ft-1, weak loss-of-function alleles (Kardailsky et al., 1999; Kobayashi et al., 1999; Robson et al., 2001), were compared (Figures 6C and 6D). GA3 was similarly effective in suppressing gi-3, co-2, and ft-1. By contrast, spy-4 suppression of gi-3, co-2, and ft-1 was progressively weaker (Figures 6C and 6D) as the mutations acted further downstream in the pathway (Figure 4B). Considering that co-2 and ft-1 are leaky alleles, these results are consistent with SPY acting upstream of CO and FT.

The levels of CO and FT RNA are reduced by gi (Suárez-Lopez et al., 2001). If SPY and GI act as a complex in the photoperiod pathway, the mechanisms of suppression of the late flowering of gi by spy may involve increasing CO and FT RNA abundance. CO and FT RNA abundance was determined in wild-type, gi-2, spy-4, and spy-4 gi-2 plants growing under 18L/6D cycles every 4 h for 24 h (Figure 8). At each time point, the CO and FT RNA levels in spy-4 gi-2 plants were elevated over gi-2 plants.

Figure 8.

Figure 8.

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spy Suppresses the Reduced CO and FT RNA Phenotype of gi.

(A) Total RNA was extracted from plants growing under 18L/6D conditions every 4 h. CO, FT, and UBQ10 cDNA were amplified by RT-PCR under semiquantitative conditions, resolved on agarose gels, and detected by hybridization. The experiment was repeated a second time with similar results. In the experiment shown, the 0 h time point from the gi-2 spy plants did not yield PCR products.

(B) and (C) The autoradiograms shown in (A) were scanned, and the ratio of CO or FT cDNA to UBQ10 cDNA in samples Columbia (closed circles), spy-4 (closed squares) gi-2 (open squares), and spy-4 gi-2 (open circles) were determined.

SPY Affects the Period of the Free-Running Rhythm in Cotyledon Movement

The cotyledon movement of Arabidopsis seedlings has been shown to be under the circadian control (Millar et al., 1995a). gi mutations caused alterations in the period and amplitude of the movement. The free-running period and amplitude of cotyledon movement were determined in wild-type, gi, spy, and gi-2 spy-4 seedlings and seedlings overexpressing SPY under the control of the 35S promoter of Cauliflower mosaic virus (Swain et al., 2001) (Figure 9, Table 1). As was observed previously (Park et al., 1999), all gi alleles greatly reduced the amplitude of the rhythm. Although spy had no effect on the amplitude of the rhythm, the period was lengthened in each of the three spy alleles and reduced in seedlings that overexpress SPY. The reduction in the amplitude of the rhythm of gi plants was not suppressed by spy-4. To determine if the defect in spy and gi plants was specific to plants entrained with light/dark cycles, cotyledon movements were also monitored in plant entrained using temperature cycles. For all of the genotypes, the results were similar to those of plant entrained using light/dark cycles.

Figure 9.

Figure 9.

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spy Loss-of-Function and Gain-of-Function Seedlings Display an Altered Circadian Period in Cotyledon Movement Rhythm.

(A) to (C) Cotyledon movement after entrainment by light-dark cycles. Seedlings were grown for 5 d in 12 h light/12 h dark, transferred to 24-well plates, and released into continuous light (LL).

(D) to (F) Cotyledon movement after entrainment by temperature cycles. Seedlings were grown for 7 d in 12 h 22°C/12 h 12°C, transferred to 24-well plates, and released into continuous conditions at 22°C. Cotyledon movement was recorded for 7 d. Each trace is the average of 6 to 12 individual seedlings. The experiment was repeated three times with similar results. Hatched bars indicate subjective night.

(A) and (D) spy-4 (open squares) shows a long period in cotyledon movement relative to its wild-type Columbia (closed circles).

(B) and (E) Overexpression of SPY (open squares) caused a period shortening of cotyledon movement.

(C) and (F) Loss of SPY function further decreased the amplitude and rhythmicity of a gi mutant. Cotyledon movement in spy-4 gi-2 double mutant seedlings (open diamonds) dampened more rapidly than gi-2 single mutant seedlings (open squares).

Table 1.

Alterations of Circadian Period in SPY Loss-of-Function and Gain-of-Function Seedlings

Condition Genotype Period RAEa Amplitude N
Light entrainment Col 24.52 ± 0.14 0.12 ± 0.006 1.26 ± 0.12 12
spy-3 25.38b ± 0.27 0.22 ± 0.023 1.45 ± 0.12 12
Ler 24.18 ± 0.16 0.16 ± 0.010 1.25 ± 0.09 9
spy-5 25.19b ± 0.15 0.19 ± 0.020 1.25 ± 0.18 9
Col 24.54 ± 0.12 0.22 ± 0.015 0.97 ± 0.24 4
spy-4 26.21b ± 0.31 0.28 ± 0.034 1.37 ± 0.35 7
35S-SPY 22.91b ± 0.24 0.24 ± 0.012 2.3b ± 0.32 11
gi-2c 24.26 0.08 0.60
spy-4 gi-2c 27.42 0.35 0.54
gi-1c 22.29 0.12 0.90
gi-3c 21.24 0.25 0.45
gi-4c 19.44 0.09 0.38
gi-5c 19.53 0.18 0.63
gi-6c 18.66 0.17 0.43
Temperature entrainment Col 24.34 ± 0.15 0.27 ± 0.036 1.01 ± 0.18 15
spy-4 26.53b ± 0.48 0.27 ± 0.028 1.21 ± 0.12 10
35S-SPY 22.70b ± 0.39 0.19 ± 0.010 2.29b ± 0.52 4
gi-2c 25.32 0.14 0.55
gi-2 spy-4c 26.95 0.20 0.55
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Seedlings were grown under entraining conditions for 5 to 7 d before being transferred to 24-well cloning plates in constant light (30 to 40 μmol s−1 m−2) and temperature (22°C) for data collection. All numbers are provided ± se.

a

Relative amplitude error.

b

Statistically different from the respective wild type by Student's t test (P < 0.01).

c

The low amplitude of these genotypes did not allow for an accurate period estimate; therefore, the circadian parameters of the average trace are given as an indication.

DISCUSSION

SPY And GI Interact Physically

SPY and GI interacted in three different assays (Figures 1 and 2). Phenotypic analyses of mutants indicate that these proteins function in common signaling pathways suggesting that physical interaction between GI and SPY is critical to their role in at least some pathways. Our preliminary attempts to identify a SPY/GI complex in plants, however, have not been successful. This lack of success may be because of the very low abundance of both proteins. Although the existence of a SPY/GI complex has not been demonstrated in planta, these proteins are expected to have opportunities to interact because both are nuclear localized and SPY is expressed throughout the plant (Huq et al., 2000; Swain et al., 2002).

The N-terminal half of SPY has 10 tandem TPRs. The spy-2 mutation deletes portions of TPRs 8 and 9. We have shown previously (Tseng et al., 2001) that the TPR domain of SPY interacts with itself. When the strength of spy-2 TPR interactions with wild-type SPY TPRs and GI was examined using the yeast two-hybrid system (Figure 1C), the spy-2 mutation weakened the TPR–TPR interaction and strengthened the interaction with GI. The decreased TPR–TPR interaction may explain why spy-2 is a strong allele even though it is predicted to express the full-length protein. Rat OGT has 11 TPRs and is a trimeric protein. Deletion of the first six TPRs of rat OGT blocks multimerization (Kreppel and Hart, 1999). Therefore, it is possible that the spy-2 protein is inactive because it does not assemble into trimers. In addition to exhibiting a spy phenotype, spy-2 plants also have phenotypes that are not observed with other alleles (Jacobsen and Olszewski, 1993). One explanation for the novel spy-2 phenotypes is that the mutation differentially affects interactions with other proteins. This is supported by the observation that spy-2 increases the interaction with GI. An alternative explanation for the effect of the spy-2 mutation on the interaction with GI in yeast is that it disrupts spy-2 multimerization thereby making more spy-2 available for interaction with GI.

GI and SPY Function in Common Pathways

To gain insight into the role of the SPY-GI interaction in Arabidopsis, several different phenotypes known to be affected in gi were examined in single and double mutants. Consistent with SPY and GI acting in common pathways, loss of SPY function suppresses loss of GI function phenotypes, including the long hypocotyl phenotype of plants grown under red light (Figure 3), late flowering (Figure 6), and reduced CO and FT RNA abundance (Figure 8).

The strongest suppression of gi-2 by spy-4 was observed for hypocotyl length. When grown under red light, gi-2 seedlings have a long hypocotyl (Huq et al., 2000; Figure 3). The hypocotyl length of both spy-4 and spy-4 gi-2 seedlings was indistinguishable from the wild type. By contrast, spy-4 partially suppressed the late flowering and reduced CO and FT RNA levels of gi-2.

GI affects flowering time by acting in the long-day pathway (Araki and Komeda, 1993; Koornneef et al., 1998; Fowler et al., 1999). SPY is believed to delay flowering by inhibiting the GA pathway (Mouradov et al., 2002; Simpson and Dean, 2002). Consistent with SPY affecting flowering by also acting in the long-day pathway, spy-4 suppressed the late flowering of gi-2 and gi-3 (Figure 6) more effectively than daily application of 5 × 10−4 M GA3, a treatment that suppresses ga1, a mutation blocking GA synthesis (Sun and Kamiya, 1994), more effectively than spy-4 (Figure 7).

GI acts upstream of CO and FT in the long-day pathway and is required for the accumulation of CO and FT RNA. spy-4 suppresses the reduction of CO and FT RNA in gi-2 plants, indicating that SPY functions in the long-day pathway upstream of CO and FT. Also consistent with SPY acting upstream of CO and FT, spy-4 suppressed the late flowering of gi-3 more effectively than that of co-2 and ft-1 (Figures 6C and 6D). The greater suppression of co-2 and ft-1 by spy-4 than treatment with GA3 would be inconsistent with SPY acting upstream of CO and FT if the alleles used in these experiments were null. However, co-2 and ft-1 are leaky alleles (Kardailsky et al., 1999; Kobayashi et al., 1999; Robson et al., 2001). Although ft-1 is a weak allele, GA3 and spy-4 were similarly effective suppressors of it, suggesting that spy-4 suppression of ft-1 is primarily by activation of the GA pathway and, furthermore, that in the long-day pathway ft-1 is epistatic to spy-4.

The suppression of the late flowering and CO and FT RNA accumulation defects of gi plants is consistent with the model that GI is a negative regulator of SPY (Figure 4A). This model predicts that, relative to the wild type, spy-4 plants will have elevated levels of CO RNA. Consistent with this prediction, at five of seven time points examined, spy-4 had more CO RNA than the corresponding wild-type plants (Figures 8A and 8B). The model also predicts a similar effect on FT RNA, which was only observed at three time points. Although we do not know why FT RNA was not elevated at more time points in spy-4, one possibility is that FT RNA levels are maximal in wild-type plants and cannot be elevated further.

Whereas spy-4 did not suppress the gi-2 defect in the circadian rhythm in cotyledon movements, spy-4 plants and plants overexpressing SPY exhibited defects consistent with SPY functioning in this process (Figure 9, Table 1). The free-running period was lengthened in spy-3, spy-4, and spy-5 plants and reduced in plants overexpressing SPY under the control of the 35S promoter of Cauliflower mosaic virus. Although all six gi alleles examined greatly reduced the amplitude of the rhythm, which prevents accurately determining the free-running period, the period of the rhythm was shortened by the majority of the gi alleles. The amplitude of the rhythm was not affected by any of the spy alleles. Whereas the period of spy-4 gi-2 seedlings was longer than that of gi-2 plants, the period of gi-2 was similar to the wild type. The reduced amplitude of gi-2 was not suppressed in the spy-4 gi-2 double mutants. The failure of spy to suppress dampening of the rhythm in cotyledon movement is surprising because spy-4 has been shown to suppress the dampening of the rhythm in transpiration by gi-2 (Sothern et al., 2002). When grown under 14L/10D cycles, the amplitude of the transpiration rhythm in the wild type and gi-2 was similar and greater than that of spy-4 and spy-4 gi-2 plants. Upon transfer from 14L/10D cycles to continuous light conditions, the amplitude of transpiration rhythm rapidly damped in gi-2 but damped less dramatically in wild-type, spy-4, and spy-4 gi-2 plants. The results of these two studies suggest that SPY and GI play different roles in the rhythms in cotyledon movement and transpiration. Alternatively, the amplitude of the rhythm in transpiration is too low in spy-4 and spy-4 gi-2 plants growing under 14L/10D cycles (entraining conditions) for additional changes in amplitude to be detected when the plants are transferred to continuous light (free-running conditions). Similar to what was observed for cotyledon movement, the period of the free-running rhythm in transpiration is increased in spy-4 plants.

Although these results do not indicate whether SPY acts in input pathways, the oscillator or output pathways of the circadian clock, SPY clearly plays a role in two processes that are controlled by the circadian clock, cotyledon movement and transpiration, suggesting that if SPY functions in output pathway(s), it is acting in a part that is common to both responses. The effects of spy-4 mutations and SPY overexpression on the free-running period of the rhythm in cotyledon movement occurred when the seedlings were entrained with either light/dark cycles or with temperature cycles (Figure 9, Table 1), indicating that SPY action is not restricted to the light input pathway alone and suggesting that if SPY affects input pathway(s), it is acting in a portion that is common to both temperature and light inputs. These data are also consistent with SPY playing a role in the central oscillator. To distinguish between these different possible roles for SPY, we are currently investigating the expression of the clock genes CCA1, LHY, and TOC1 in the spy-4 and SPY overexpressing plants.

SPY Acts in Two Pathways Controlling Hypocotyl Elongation

The effects of spy on hypocotyl length are complex. spy decreases hypocotyl length in the dark and increases it under far-red light. Although spy has no effect under red light, it suppresses the red-light phenotype of gi (Figure 3). To explain these complex results, we propose that SPY has two roles in hypocotyl elongation (Figure 4A). SPY inhibits hypocotyl elongation by negatively regulating the response to GA. In addition, it is a promoter of elongation. Furthermore, we propose that the positive role of SPY, but not the negative role, is inhibited by red light acting through GI.

The model predicts that in the dark the positive role of SPY in hypocotyl elongation will be maximal because it is not being inhibited by red light (Figure 4A). By contrast, because the GA response of a dark-grown hypocotyl is saturated (Gendreau et al., 1999; Swain et al., 2001), the negative role of SPY in the GA pathway will be minimal. These predictions are consistent with the short hypocotyl phenotype of spy growing in darkness (Figure 3).

Inhibition of hypocotyl elongation by both red and far-red light involves modulation of GA signaling; however, the mechanisms of this modulation are different. Exposure of etiolated seedlings to red light reduces both GA levels and responsiveness to GA (Reed et al., 1996; Reid et al., 2002). By contrast, far-red light only decreases the concentration of bioactive GAs (Toyomasu et al., 1993; Reid et al., 2002). Under red light, spy has no detectable effect on the hypocotyl length (Figure 3), suggesting that the positive and negative roles of SPY in hypocotyl elongation are equivalent. The hypocotyls of gi-2 plants are predicted to be long under red light because the positive role of SPY in elongation is no longer inhibited. Consistent with this model, spy-4 suppresses gi-2.

Because far-red light reduces GA levels, SPY's negative regulation of hypocotyl elongation is more dominant under far-red light than it is under other light conditions. Consistent with this prediction, spy-4 has a long hypocotyl phenotype under far-red light. This model predicts that if the negative effect of SPY in GA signaling is overcome by growing seedlings in the presence of high doses of GA3, spy-4 hypocotyls will be shorter than the wild type because of the loss of SPY's positive role in hypocotyl growth (Figure 4A). When this prediction was tested by growing the wild type and spy-4 on different concentrations of GA3 (Figure 5), the magnitude of the spy-4 long hypocotyl phenotype decreased with increasing GA3, and at 1 × 10−4 M GA3, the hypocotyl length of the wild type exceeded that of the spy-4.

Whereas GI and SPY participate in the regulation of hypocotyl length by red light, the hypocotyls of spy and gi seedlings respond to red light (Figure 3). Therefore, SPY and GI must modulate the activity of the pathway, be functionally redundant with other Arabidopsis proteins, or only function in one of several pathways mediating red light inhibition of hypocotyl elongation. Red light affects hypocotyl elongation both by triggering photomorphogenesis and by affecting the circadian clock (McClung et al., 2002). Because GI and SPY affect the circadian clock, it is possible that they influence hypocotyl elongation by affecting the circadian clock and not photomorphogenesis.

SPY May Function Independently of GI

Although there is evidence that GI and SPY function in common pathways, there also is evidence that GI plays no role in GA responses, suggesting that SPY can act independently of GI. The hypocotyls of gi and wild type grown under far-red light have similar GA dose–response curves (Figure 5), indicating that loss of GI function does not alter the sensitivity or response of the hypocotyl to GA. By contrast and consistent with SPY functioning as a negative regulator of GA response, the response curves for both spy and gi-2 spy seedlings plateau at a lower concentration of GA3 than the wild type. We are not aware of any evidence suggesting that GI functions independently of SPY.

Conclusions

The data presented here show that SPY and GI can interact physically and that they function in common pathways. Data are also presented that GI is a light-dependent negative regulator of SPY. GI is not similar to any protein with a known function (Fowler et al., 1999; Park et al., 1999; Huq et al., 2000), and the mechanism by which GI inhibits SPY is unknown. In view of the interaction between SPY and GI in yeast and in vitro (Figures 1 and 2), the negative regulation of SPY by GI may involve direct interaction between these proteins. Because SPY is believed to be an O-GlcNAc transferase (Thornton et al., 1999a), GI is likely acting by inhibiting the activity of SPY toward specific substrates. The resulting changes in the extent of O-GlcNAc modification of substrate proteins could affect the activity, stability, or localization of these proteins. Because the circadian clock is involved in flowering and hypocotyl elongation (McClung et al., 2002), it will be interesting to learn if any of the components of the circadian clock are modified by SPY and if this modification is regulated by GI.

METHODS

Yeast Two-Hybrid Screening

Plasmids expressing SPY bait and prey proteins were constructed in pAS1-CYH2 and pACT II as described previously (Tseng et al., 2001). Plasmids expressing prey protein were prepared from an Arabidopsis thaliana λ-ACT cDNA expression library (Kim et al., 1997) obtained from the ABRC (Columbus, Ohio; stock number CD4-22). Yeast transformation and library screenings were conducted as described by Bai and Elledge (1997).

To test the specificity of the interaction, plasmid DNA from interacting clones was rescued and retransformed into the yeast cell line Y190 expressing a bait protein consisting of the TPR domain of Chlamydomonas reinhardtii Mbb1. The plasmid expressing Mbb1 as bait was constructed by subcloning the BamHI to SalI fragment from an Mbb1 cDNA clone (Vaistij et al., 2000) into the corresponding sites of pAS1-CYH2.

A plasmid expressing GI as bait, pDB-GI, was constructed by subcloning the GI coding region from a GI prey clone identified in the original two-hybrid screen into pAS1-CYH2. The GI coding region was PCR amplified using the primers 5′-TCGACTGCAGCCCAAAGTTGTGCC-3′ and 5′-GGGTGCCTGCAGTTGGGACAAGGA-3′, which generated PstI sites that were used for the subcloning.

To introduce the spy-2 mutation into the bait-TPR, SPY cDNA was amplified in separate reactions with primer sets of 5′-TTTCCCCTGATAACACTCCACAGCT-3′ plus BINF, 5′-AGTTACAAAAAAATGGTGGGACTGGAAGATG-3′ and 5′-TATCAGGGGAAAATGGATGCTG-3′ plus JP58, 5′-GAGATCCAGCCATTAGAT-3′, for 20 cycles as described (Tseng et al., 2001). Equal amounts of both PCR products were combined and reamplified with the BINF and JP58 primers at annealing temperature of 40°C for 20 cycles. The reamplified products were digested with PstI and BamHI, then subcloned to the pAS-TPR plasmid digested with PstI and BamHI.

In Vitro Protein Interaction Assay

To test the physiological association of SPY's TPR and GI, in vitro binding assays were conducted using the TPR domain of SPY expressed as a fusion protein with the bacterial MBP (New England Biolabs, Beverly, MA) as described previously (Tseng et al., 2001).

Escherichia coli Pull-Down Experiments

The SPY open reading frame was PCR amplified using the primers 5′-ACCCGACGTCATGGTGGGACTGGAAGATGA-3′ and 5′-TTTGCGGCCGCCAGCACGTCGATTTGCTGGT-3′, primers that incorporated AatII and NotI sites at the 5′- and 3′-ends of the open reading frame, respectively. PCR was performed in a 50-μL reaction containing 7.5 ng of a SacI-linearized SPY cDNA clone in pBluescript KS− with Pfx DNA polymerase as recommended by the supplier (Invitrogen, Carlsbad, CA). The PCR product was digested with AatII and NotI and cloned into pETCOCO-1 (Novagen, Madison, WI). The construct was sequenced to confirm that it was correct. The resulting construct, pETCOCO:SPY, expresses a His-tag:S-tag:SPY fusion protein under the control of the T7 RNA polymerase promoter. The plasmid pMAL-GI, which expresses an MBP-GI fusion protein, was produced by subcloning a PstI fragment from pDB-GI into pMAL-c2 (New England Biolabs).

The E. coli strain OragamiB (Novagen) was cotransformed with pETCOCO:SPY and pMAL-c2 or pMAL-GI. The resulting strains were grown in LB medium containing 1% (w/v) glucose, 15 μg/mL of chloramphenicol, and 75 μg/mL of ampicillin. For protein expression, cultures were grown to an OD600 of 0.5 at 25°C, isopropylthio-β-galactoside was added to 1 mM, and the culture was grown for an additional 2 h. The cells were then collected by centrifugation, and the pellet was stored at −20°C. Cell were resuspended in 6 mL of 25 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 0.1% Triton X-100, and 1× Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) and lysed using a French press. The lysate was cleared by centrifugation at 12,000 rpm in a JA20 rotor for 15 min. After centrifugation, 6 mL of 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM NaN3, 10 mM β-mercaptoethanol, and 30 μL of amylose resin (New England Biolabs) were added. After agitation at 4°C for 2 h, the amylose resin and associated MBP proteins were collected by centrifugation at 2000 rpm in a JA20 rotor for 30 s. The pelleted resin was transferred to a 1.5-mL microfuge tube and washed three times with 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM NaN3, and 10 mM β-mercaptoethanol. The washed pellet was resuspended in 4× SDS loading buffer and boiled for 2 min. The proteins released from the resin were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. S-tagged proteins were detected using an S-tag antibody conjugated with horseradish peroxidase as recommended by the supplier (Novagen). The blot was stripped and then probed with anti-MBP monoclonal antibody as recommended by the supplier (New England Biolabs). Bound antibody was detected with protein A conjugated with horseradish peroxidase (Sigma, St. Louis, MO).

Measurement of Hypocotyl Length of Seedlings Grown under Different Light Conditions

The Arabidopsis gi-2 mutant was obtained from the ABRC. The spy-4 mutation was backcrossed into the Columbia background six times (Swain et al., 2001). Seeds were surface sterilized (Tseng et al., 2001) and grown according to Huq et al. (2000). In some experiments, GA3 (Sigma) was included in the growth medium. The fluence rates were monitored using a LiCor-1800 spectroradiometer (LiCor, Lincoln, NE). Seedlings were germinated in the dark (at 21°C) or under continuous red light (4.49 μmol m−2 s−1 at 21°C) or continuous far-red light (6.6 μmol m−2 s−1 at 24°C) for 6 d. gi and wild-type seedlings grown under similar conditions have been previously shown to exhibit differences in hypocotyl elongation (Huq et al., 2000). Hypocotyl length was measured under a dissecting microscope with an ocular micrometer. Similar results were obtained in two independent experiments.

Flowering Time Measurement

The F1 seeds of the crosses between spy-4 and gi-3, co-2, and ft-1, all in the Ler background, were a gift from S. Jacobsen. F1 seeds were also generated by crossing spy-4 in the Columbia background (see above) and gi-2. It was possible to identify the spy-4 gi-3, spy-4 co-2, spy-4 ft-1, and spy-4 gi-2 double mutants in the F2 populations because they germinated in the presence of paclobutrazol (35 mg/L) and flowered later than the other plants selected under these conditions. For determination of flowering time, plants were grown in a growth chamber under long-day conditions (16L/8D) at 22 to 23°C. The rosette leaf, cauline leaf number, and the number of days to flowering were recorded when petals of the first flower were visible. Although both the leaf number and days to flowering showed similar trends, only the leaf number data are presented. For GA treatment, the GA3 solution was replenished from a GA3 stock solution of 10 mg/mL (in 95% ethanol) every 2 d. Separate plants grown under the long-day conditions were sprayed with 5 × 10−4 M of GA3 everyday. Control plants were sprayed with 1.6% of ethanol everyday.

Semiquantitative RT-PCR

Tissues of 2-week-old plants growing under long-day conditions (18L/6D) at 22°C were harvested approximately every 4 h for 24 h after dawn. Total RNA was extracted with TRI reagent (Molecular Research Center, Cincinnati, OH) and treated with RNase-free DNase I (Promega, Madison, WI). Approximately 2 μg of total RNA were used to synthesize the first-strand cDNA with SuperScript first-strand synthesis system for RT-PCR (Invitrogen) following the manufacturer's instructions. PCR was performed according to Klimyuk et al. (1993). In the same reaction tube, first-strand cDNAs were amplified with a UBQ10 primer pair (5′-AACTTTCTCTCAATTCTCTCTACC-3′ and 5′-CTTCTTAAGCATAACAGAGACGAG-3′) (Debeaujon et al., 2001) and either CO primers (5′-ATGTTGAAACAAGAGAGTAACG-3′ and 5′-GTTATGGTTAATGGAACCATTG-3′) or FT primers (5′-GATATCCCTGCTACAACTGG-3′ and 5′-TAGATGCATAAATCTCATCAGAG-3′). Under the conditions used, production of all PCR products was found to increase linearly between cycles 20 and 35 (data not shown); therefore, the PCR products were quantitated after 25 cycles. PCR products were separated in 0.8% agarose gel, then transferred to nylon membranes (Osmonics, Westborough, MA). The membranes were probed with 32P-labeled UBQ10, CO, and FT probes separately. The autoradiograms were scanned, and signal intensities were quantitated using NIH Image software.

Cotyledon Movement Assay

Assessment of rhythmicity in cotyledon movement was performed as described (Millar et al., 1995a; Salomé et al., 2002). Seeds were surface sterilized by the vapor-phase method (Clough and Bent, 1998) and then plated on MS salts + 2% sucrose. For light entrainment, seedlings were grown under white light (70 to 80 μmol m−2 s−1) for 5 d in a 12-h-light/12-h-dark photoperiod. For temperature entrainment, seedlings were grown under white light (30 to 40 μmol m−2 s−1) for 7 d in a 12-h 22°C/12-h 12°C temperature regime. On the fifth or seventh day, seedlings were transferred to 24-well cloning plates (Greiner Labortechnik, Frickenhausen, Germany), and the plates were released into continuous white light (30 to 40 μmol m−2 s−1) and constant temperature of 22°C. Cotyledon movement was recorded every 20 min over 7 d by Panasonic CCTV cameras (Model WV-BP120; Matsushita Communications Industrial, Laguna, Philippines). Post-run analysis was performed using the Kujata software program (Millar et al., 1995a, 1995b), and traces were analyzed by fast-Fourier transform-nonlinear least squares (Plautz et al., 1997).

Acknowledgments

We thank G. Engelen-Eigles, G. Gardner, and M. Ni for the assistance with light experiments and David Marks, S. Swain, and L. Hartweck for critical reading of the manuscript. This research was supported by grants from the National Science Foundation (MCB-9604126, MCB-9983583, and MCB-0112826) and BARD, the United-Israel Binational Agricultural Research and Development Fund, (IS-2837-97) to N.E.O and National Science Foundation (MCB-0091008) to C.R.M.

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: Neil E. Olszewski (neil@biosci.cbs.umn.edu).

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.019224.

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