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A Gain-of-Function Mutation in IAA7/AXR2 Confers Late Flowering under Short-day Light in ArabidopsisF
Article first published online: 9 JUN 2011
© 2011 Institute of Botany, Chinese Academy of Sciences
Journal of Integrative Plant Biology
Volume 53, Issue 6, pages 480–492, June 2011
How to Cite
Mai, Y.-X., Wang, L. and Yang, H.-Q. (2011), A Gain-of-Function Mutation in IAA7/AXR2 Confers Late Flowering under Short-day Light in Arabidopsis. Journal of Integrative Plant Biology, 53: 480–492. doi: 10.1111/j.1744-7909.2011.01050.x
- Issue published online: 9 JUN 2011
- Article first published online: 9 JUN 2011
- Accepted manuscript online: 12 MAY 2011 05:04AM EST
- Received 14 Jan. 2011 Accepted 25 Apr. 2011
- GA 20-oxidases;
- Top of page
- Materials and Methods
- Supporting Information
Floral initiation is a major step in the life cycle of plants, which is influenced by photoperiod, temperature, and phytohormones, such as gibberellins (GAs). It is known that GAs promote floral initiation under short-day light conditions (SDs) by regulating the floral meristem-identity gene LEAFY (LFY) and the flowering-time gene SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). We have defined the role of the auxin signaling component INDOLE-3-ACETIC ACID 7 (IAA7)/AUXIN RESISTANT 2 (AXR2) in the regulation of flowering time in Arabidopsis thaliana. We demonstrate that the gain-of-function mutant of IAA7/AXR2, axr2-1, flowers late under SDs. The exogenous application of GAs rescued the late flowering phenotype of axr2-1 plants. The expression of the GA20 oxidase (GA20ox) genes, GA20ox1 and GA20ox2, was reduced in axr2-1 plants, and the levels of both LFY and SOC1 transcripts were reduced in axr2-1 mutants under SDs. Furthermore, the overexpression of SOC1 or LFY in axr2-1 mutants rescued the late flowering phenotype under SDs. Our results suggest that IAA7/AXR2 might act to inhibit the timing of floral transition under SDs, at least in part, by negatively regulating the expressions of the GA20ox1 and GA20ox2 genes.
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- Materials and Methods
- Supporting Information
The most dramatic phase change that flowering plants undergo is the transition from vegetative to reproductive growth. In Arabidopsis, flowering is initiated via four main genetic pathways: gibberellins (GAs), autonomous, vernalization, and light-dependent pathways. Interestingly, all these pathways appear to interact in a complex manner and converge on the transcriptional regulation of the floral integrator genes FLOWERING LOCUST, LEAFY (LFY), and the flowering-time gene SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) that act on the floral meristem-identity genes to initiate flowering (Moon et al. 2003; Boss et al. 2004; Corbesier and Coupland 2006).
Auxin controls an impressive variety of plant developmental and physiological processes, including embryonic development (Friml et al. 2003), lateral root formation (Okushima et al. 2007), tropic responses (Blakeslee et al. 2004), apical dominance (Thimann and Skoog 1934; Panigrahi and Audus 1966), and vascular development (Mattsson et al. 1999). The Arabidopsis genome encodes 29 AUXIN (AUX)/INDOLE-3–ACETIC ACID (IAA) genes that are rapidly and specifically induced in response to auxin (Abel and Theologis 1996). The protein products of these genes are quickly degraded with increasing levels of auxin in Arabidopsis (Gray et al. 2001; Zenser et al. 2001; Overvoorde et al. 2005; Dreher et al. 2006). Thus the AUX/IAA family is generally thought to act as a repressor of auxin-induced gene expression (Ulmasov et al. 1997b). The auxin-responsive degradation of AUX/IAAs requires amino acids within domain II, one of the four conserved domains found in most AUX/IAA proteins. Several auxin-insensitive dominant and semidominant mutants in AUX/IAA that have amino acid substitutions in domain II disrupt auxin responses and often result in dramatic phenotypes (Reed 2001). In Arabidopsis, there are 23 Auxin Response Factors (ARF) proteins (Okushima et al. 2005), which bind specifically to the auxin-responsive element (AUXRE)TGTCTC to regulate the expression of auxin-responsive genes (Abel and Theologis 1996; Ulmasov et al. 1997b). Transport Inhibitor Response 1 (TIR1) is a member of a small gene family that contains 5 additional Auxin Signaling F-Box Protein (AFB) proteins. Previous studies have shown that TIR1, and at least AFB1, AFB2, and AFB3, all function as auxin receptors (Dharmasiri et al. 2005a; Dharmasiri et al. 2005b; Kepinski and Leyser 2005). Perceived by the F-box protein subunit of the Skp1/Cul1/F-box Complex (SCF)TIR1/AFBs ubiquitin ligase, auxin directly promotes the recruitment of AUX/IAAs for ubiquitination, thereby derepressing the ARF transcription factors. Once freed from the AUX/IAAs, these ARFs regulate the expression of auxin-responsive genes (Lau et al. 2008).
The phytohormone GA is known to play a prominent role in regulating the timing of floral transition. Exogenous treatment with GA accelerates flowering in Arabidopsis, particularly under short-day light conditions SDs (Langridge 1957; Bagnall 1992). In addition, the mutations deficient in GA biosynthesis or impaired in GA responsiveness show alterations in flowering time (Wilson et al. 1992; Jacobsen and Olszewski 1993). Severely GA-deficient mutant ga1–3 plants, for example, are dwarfed and fail to flower under SDs. These plants treated with GA restore normal growth and flowering time (Wilson et al. 1992). Mutations that block GA responsiveness (e.g. sly1) cause delayed flowering and are insensitive to GA (Dill et al. 2004). However, mutants with increased GA signaling (e.g. rga, gai, and spy) flower early (Jacobsen and Olszewski 1993; Dill and Sun 2001).
The GA pathway regulates the timing of floral initiation, mostly through the upregulation of the floral integrators LFY and SOC1 (Corbesier and Coupland 2006). Under SDs, the flowering time of GA-biosynthetic and -signaling mutants is well correlated with the level of LFY or SOC1 expression (Blazquez et al. 1998; Moon et al. 2003). The overexpression of either of these integrators can partially rescue the non-flowering phenotype of ga1–3 (Blazquez et al. 1998; Moon et al. 2003). Previous studies have indicated that auxin and GAs overlap in their regulation of multiple aspects of plant development, including root growth and organ expansion (Fu and Harberd 2003; Frigerio et al. 2006). We demonstrate that the gain-of-function mutant of auxin signaling component IAA7/AXR2, axr2-1, flowers late under SDs. Through molecular, genetic, and physiological analyses, we demonstrate that IAA7/AXR2 is likely to be involved in the suppression of floral initiation under SDs, at least partly, in a GA-dependent manner.
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- Materials and Methods
- Supporting Information
axr2-1 mutant plants flower late under SDs
Dominant mutations in several AUX/IAA genes confer pleiotropic, auxin-related phenotypes (Liscum and Reed 2002). Furthermore, mutations in domain II that stabilize AUX/IAA proteins result in the inhibition of auxin-responsive reporter gene expression (Ouellet et al. 2001; Oono et al. 2002; Tatematsu et al. 2004). The axr2-1 mutation changes a single amino acid (Pro to Ser at codon 87) in conserved domain II of IAA7/AXR2; this stabilizes the AUX/IAA protein IAA7/AXR2, and thereby decreases auxin response (Nagpal et al. 2000). We used axr2-1 mutants to explore whether auxin signaling might regulate floral transition. We analyzed the flowering-time phenotype of wild-type (WT) and axr2-1 mutant plants in long-day (LDs) and SDs photoperiods illuminated by white light. We found that axr2-1 mutant plants flowered at approximately the same time as WT plants under LDs (Figure 1A and Table S2). However, under SDs, axr2-1 mutant plants flowered significantly later than the WT, as indicated by days to flowering and the number of rosette leaves at flowering (Figure 1B–D).
Transgenic plants overexpressing indoleacetic acid lysine synthetase show a late flowering phenotype under SDs
It has been demonstrated that indoleacetic acid lysine synthetase (iaaL) in Pseudomonas syringae subsp. savastanoi is able to catalyze the conjugation process of free IAA with lysine and produce IAA lysine. The expression of iaaL in transgenic plants reduces endogenous free IAA levels and results in auxin-deficient phenotypes (Romano et al. 1991; Spena et al. 1991). In an attempt to characterize the role of auxin in the regulation of floral initiation, we employed a constitutive 35S promoter to drive the hemagglutinin (HA) epitope-tagged iaaL, and transformed it into WT Arabidopsis plants. We obtained more than 30 independent transgenic lines, approximately 20 of which displayed a decreased apical dominance phenotype. Transgenic 35Spro iaaL HA plants were analyzed by Western blot using an antibody against HA. Our results indicated that they accumulated varied levels of iaaL (Figure 2A). We then analyzed the flowering-time phenotype of three independent homozygous transgenic plants under LDs and SDs. All these transgenic lines flowered at approximately the same time as the WT under LDs (Figure 2B and Table S2), whereas they flowered significantly later than the WT under SDs, as indicated by days to flowering and the number of rosette leaves at flowering (Figure 2C–E). Moreover, the severity of the late flowering-time phenotype is positively correlated with iaaL HA protein levels. These data therefore suggest that auxin might act positively in regulating floral induction under SDs.
Exogenous application of GA3 rescues the late flowering phenotype of axr2-1 plants
Mutations that affect GA biosynthesis or signal transduction delay Arabidopsis flowering weakly under LDs, but severely under SDs. The GA biosynthesis-deficient mutant ga1-3 never flowers under SDs, but flowers when treated with exogenous GA3 (Sun et al. 1992; Wilson et al. 1992). The gai mutation affects GA signaling, and the gai mutant plants do not respond to exogenously-applied GA3 (Peng et al. 1997). To explore whether GA biosynthesis and/or signaling might be impaired in axr2-1 plants, we applied exogenous GA3 to these plants and analyzed the flowering phenotype under SDs. As shown in Figure 3 and Table S2, the GA3-treated plants of axr2-1 flowered almost as early as the those of the WT, as indicated by days to flowering and the number of rosette leaves at flowering. These data indicate that GA biosynthesis is probably impaired in axr2-1 plants.
SPINDLY genetically acts downstream of IAA7 to regulate flowering time
The Arabidopsis SPINDLY (SPY) protein negatively regulates GA signaling, and the loss-of-function SPY mutation can suppress the late flowering phenotype of the GA-deficient ga1 mutant (Jacobsen and Olszewski 1993; Silverstone et al. 2007). To examine whether SPY genetically interacts with IAA7 in regulating flowering time, we generated axr2-1 spy-1 and axr2-1 spy-3 double mutants. We found that the flowering phenotype of these double mutants was similar to that of spy-1 and spy-3 single mutants under SDs and LDs (see Figure S1 and Table S2). These results indicate that the SPY mutation is epistatic to the IAA7 mutation.
Levels of LFY and SOC1 transcripts are reduced in axr2-1 mutant plants
It has been demonstrated that GA promotes flowering of Arabidopsis plants by activating the floral meristem-identity genes LFY and SOC1 (Corbesier and Coupland 2006; Roux et al. 2006). To explore whether the late flowering phenotype observed for the axr2-1 mutant is correlated with the reduced expression of LFY and SOC1, we examined the LFY and SOC1 transcript levels in axr2-1 mutant plants by quantitative real-time polymerase chain reaction (qRT PCR). We examined the LFY and SOC1 transcript levels at the time when the WT plants flowered, as previously described (Achard et al. 2007). We found that the levels of both the LFY and SOC1 transcripts in the axr2-1 mutant plants were significantly lower than those in the WT plants (Figure 4A, B). However, upon exogenous GA3 treatment, the expressions of LFY and SOC1 in the axr2-1 mutant plants increased dramatically, and for unknown reasons, reached a level greater than that in WT plants (Figure 4C, D). These data suggest that the late flowering phenotype observed for axr2-1 mutant plants might result from the reduction in LFY and SOC1 expressions.
Overexpression of either SOC1 or LFY in axr2-1 mutant background accelerates flowering under SDs
Based on the finding that the expressions of SOC1 and LFY are reduced in axr2-1 mutant plants under SDs, we predicted that the overexpression of SOC1 or LFY in axr2-1 mutants would be able to rescue the late flowering phenotype of the axr2-1 mutant. To test this, we prepared a construct constitutively expressing SOC1 (35Spro SOC1) and transformed it into WT Arabidopsis. More than 20 independent transgenic plants exhibiting an early flowering phenotype were obtained, and the expression of the SOC1 transgene was examined by reverse transcription (RT) PCR (Figure 5B). We analyzed in detail the flowering phenotype of four independent homozygous lines (35Spro-SOC1#1, 35Spro SOC1#2, 35Spro-SOC1#5, and 35Spro-SOC1#8), and found that transgenic plants flowered earlier than WT plants under both LDs and SDs (Figure 5A). One representative line, 35Spro-SOC1#1, was introduced into the axr2-1 mutant background by genetic crossing. We found that the 35Spro-SOC1#1/axr2-1 plants flowered almost as early as the 35Spro-SOC1#1 plants, and much earlier than the axr2-1 mutant plants under SDs (Figure 5C, D; Table S2).
We then generated a construct constitutively expressing LFY (35Spro LFY) and transformed it into WT Arabidopsis. Since almost all the transgenic lines that exhibited an extremely early flowering phenotype failed to set seeds, we encountered major difficulties introducing 35Spro LFY into the axr2-1 mutant background by genetic crossing. In an attempt to circumvent this problem, we transformed the 35Spro LFY construct and control construct (35Spro NPTII) into WT and axr2-1, respectively. The T1 seeds were screened on Murashige and Skoog (MS) plates containing 100 mg/mL kanamycin and grown under SDs. We found that the transgenic axr2-1 mutant plants expressing 35Spro LFY flowered significantly earlier than the axr2-1 mutants under SDs (see Figure S2). These results, in conjunction with our qRT PCR analysis of SOC1 and LFY expressions (Figure 4A, B), suggest that the late flowering phenotype of the axr2-1 mutant at least partially results from the decrease in SOC1 and LFY expression.
Gibberellin 20 oxidase1 and GA20 oxidase2 transcripts are down-regulated in axr2-1 plants
The GA biosynthetic pathway is well characterized in Arabidopsis, and a number of genes encoding metabolic enzymes have been identified (Hedden and Phillips 2000). GA20 oxidases (GA20ox), for example, are major determinants of GA production and GA-dependent development in Arabidopsis (Huang et al. 1998; Coles et al. 1999). The ga20ox1 ga20ox2 double mutant flowers late (Rieu et al. 2008). Based on the finding that GA biosynthesis might be impaired in axr2-1 plants under SDs, we postulated that the expressions of the genes encoding key enzymes in the GA biosynthetic pathway might be reduced. To test this, we examined GA20ox1 and GA20ox2 transcription levels under SD-grown axr2-1 seedlings by use of RT PCR and qRT PCR. As shown in Figure 6, the GA20ox1 and GA20ox2 transcripts in axr2-1 seedlings are significantly lower than those in WT seedlings. These results suggest that the late flowering phenotype observed for axr2-1 plants under SDs is at least partly attributable to the reduced expression of GA20ox1 and GA20ox2 genes.
Flowering phenotype of ARF mutants
AUX/IAA proteins inhibit auxin responses through interactions with ARF transcription factors (Vanneste and Friml 2009). Thus, a plausible explanation for the effects of axr2-1 on flower initiation would be that axr2-1 interacts with an as yet unidentified ARF that regulates these auxin responses. We therefore analyzed the flowering time of a series of loss-of function mutants of ARF (see Table S1). These arf single mutants barely showed a flowering-time phenotype under LDs or SDs, indicating that ARFs might act redundantly in regulating flowering time. We then analyzed the flowering time of double mutants among closely-related ARF genes, such as arf3-2 arf4-1, arf6-1 arf8-2, and arf7-1 arf19-1. We observed no flowering phenotype for arf3-2 arf4-1 or arf6-1 arf8-2 mutants under either LDs or SDs (see Table S1). However, we found that the arf7-1 arf19-1 double mutant flowered significantly later than the WT under SDs in terms of days to flowering (see Table S1). With regard to the number of rosette leaves, arf7-1 arf19-1 mutant plants did not show a pronounced late flowering phenotype (see Table S2). These results suggest a possible functional redundancy among ARF genes (see Discussion).
The auxin receptor quadruple mutant plants do not show a late flowering phenotype under SDs
Previous reports indicate that TIR1/AFBs act redundantly to regulate diverse aspects of plant growth and development (Dharmasiri et al. 2005b; Cecchetti et al. 2008; Pan et al. 2009). With the demonstrations that axr2-1 plants flower late under SDs, we predicted that the loss-of-function mutant of TIR1, AFB1, AFB2, and AFB3 would exhibit a late flowering phenotype. To test this possibility, we identified and characterized T-DNA insertional mutants of afb1-5 (SALK_144884), afb2-3 (SALK 137151), and afb3-4 (SALK_068787) by PCR genotyping (see Figure S4). We further analyzed whether tir1-1, afb1-5, afb2-3, and afb3-4 mutants express their transcripts by RT PCR, and found that afb1-5, afb2-3, and afb3-4 mutants did not produce full-length transcripts, whereas the tir1 1 mutant did. However, both afb1-5 and afb3-4 mutants clearly accumulated truncated transcripts, whereas the afb2-3 mutant did not (see Figure S3). Previous reports have shown that tir1-1 is a G147D mutation, and AUX/IAA degradation is largely inhibited in this mutant (Ruegger et al. 1998; Maraschin Fdos et al. 2009). Thus, afb1 and afb3 mutants might not be null.
We analyzed the flowering phenotype of the tir1-1, afb1-5, afb2-3, and afb3-4 single mutant plants under SDs and LDs. We observed no flowering phenotype for these mutants (data not shown). To examine whether TIR1 and AFBs might function redundantly to regulate flowering time, we generated a tir1-1 afb2-3 double mutant, a tir1-1 afb2-3 afb3-4 triple mutant, and a tir1-1 afb1-5 afb2-3 afb3-4 quadruple mutant, and analyzed their flowering phenotypes under SDs and LDs. Unexpectedly, none of these mutants showed a flowering-time phenotype (see Figure S4 and Table S2). These results indicate that other auxin receptors might be involved in mediating floral initiation (see Discussion).
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- Materials and Methods
- Supporting Information
IAA7/AXR2 is involved in the inhibition of floral initiation under SDs
Several gain-of-function iaa mutants have been identified that have missense mutations in domain II. These include iaa1/axr5 (Yang et al. 2004), iaa3/shy2 (Tian and Reed 1999), iaa6/shy1 (Kim et al. 1996), iaa7/axr2 (Nagpal et al. 2000), iaa12/bdl (Hamann et al. 2002), iaa14/slr (Fukaki et al. 2002; 2005), iaa17/axr3 (Rouse et al. 1998), iaa18/crane (Uehara et al. 2008), iaa19/msg2 (Tatematsu et al. 2004), and iaa28 (Rogg et al. 2001). These gain-of-function mutations stabilize AUX/IAA proteins, thereby inducing a variety of auxin-related phenotypes (Reed 2001). The dominant axr2-1 mutation changes a single amino acid substitution within the domain II motif of IAA7/AXR2 (Pro-87-Ser) and increases the IAA7/AXR2 protein stability (Nagpal et al. 2000; Gray et al. 2001). Previous studies have shown that axr2-1 exhibits various altered auxin responses, such as shortened hypocotyls, de-etiolated phenotypes in dark, wavy leaves, reduced leaf size, and loss of gravitropism (Nagpal et al. 2000). In this study, we demonstrated that axr2-1 mutant plants flowered significantly later than the WT under SDs, as indicated by both days to flowering and the number of rosette leaves at flowering (Figure 1). Since auxin is shown to promote the degradation of AUX/IAA proteins, including IAA7 (Gray et al. 2001), it is reasonable to predict that reduction in the free IAA levels will lead to an increase in the protein stability of AUX/IAA and result in late flowering. Indeed, the transgenic plants overexpressing iaaL that show similar auxin-deficient phenotypes to those described previously (Romano et al. 1991; Zhao et al. 2001) flower significantly later than the WT under SDs (Figure 2). Therefore, these results indicate that IAA7, as well as other AUX/IAA proteins, might be involved in the regulation of flowering time under SDs, and further imply that auxin might be involved in the regulation of flowering time under SDs.
IAA7 regulates floral transition, partly via the regulation of GA20ox1 and GA20ox2 expressions
Previous reports have shown that auxin affects GA production by regulating the expression of different GA biosynthetic genes in a range of plant species (Ross et al. 2000; Wolbang and Ross 2001; Wolbang et al. 2004; Frigerio et al. 2006). Our demonstration that the late flowering phenotype of axr2-1 mutants and 35Spro-iaaL-HA transgenic plants under SDs was rescued by the exogenous application of GA implies that GA biosynthesis is impaired in these plants (Figure 3). We show that the expression of GA20ox1 and GA20ox2 was downregulated in axr2-1 and 35Spro-iaaL-HA seedlings (Figure 6). Moreover, axr2-1 plants accumulated relatively lower levels of both LFY and SOC1 transcripts than WT plants, but similar levels of LFY and SOC1 transcripts were detected in GA-treated axr2-1 and WT plants (Figure 4). Consistent with these results, the overexpression of SOC1 or LFY in the axr2-1 mutant background rescued the late flowering-time phenotype of the axr2-1 mutant under SDs (Figure 5 and Figure S2). These observations indicate that IAA7/AXR2 might regulate the timing of floral transition under SDs, at least in part, through regulating the expression of GA20ox1 and GA20ox2 genes.
How can GA20ox1 and GA20ox2 be regulated by IAA7? It is known that auxin can regulate the transcription of many genes at the cellular level (Abel and Theologis 1996). Several families of auxin-regulated genes, including members of the AUX/IAA, SMALL AUXIN-UP RNA, and Gretchen Hagen3 gene families, have been identified. They have distinct, conserved AUXREs that are typically represented by the signature sequence TGTCTC in their promoter regions (Abel and Theologis 1996). ARFs can bind specifically to AUXREs in the promoter region of auxin-response genes (Ulmasov et al. 1997a; Okushima et al. 2007). Previous reports have shown that auxin induces the expression of GA biosynthetic genes, including GA20ox (Frigerio et al. 2006). This indicates that GA20ox might be an early auxin-responsive gene. Thus, we sought to examine whether GA20ox has AUXREs, and detected two perfect TGTCTC elements and four TGTC elements in the promoter of GA20ox1, and two TGTC elements in the promoter of GA20ox2 (results not shown). It will be worth investigating in future studies whether ARFs bind directly to the promoters of GA20ox1 and GA20ox2.
Is auxin involved in regulating floral initiation under SDs?
Based on the demonstrations that axr2-1 mutant and iaaL-overexpressing plants flower very late under SDs, we predicted that auxin might act to promote floral initiation. If this prediction is true, the mutants of the auxin receptors and other auxin signaling components downstream of AUX/IAA, such as ARFs, would exhibit a flowering phenotype. However, although we observed that tir1-1 afb2-3 afb3-4 triple and tir1-1 afb1-5 afb2-3 afb3-4 quadruple mutants exhibit a variety of auxin-deficient phenotypes similar to those described previously (Parry et al. 2009), we failed to observe a late flowering phenotype for any one of the single, double, triple, or quadruple mutants of tir1-1, afb1-5, afb2-3, and afb3-4 (see Figure S4). Thus, although this might be due to the fact that some of the receptor mutants that were utilized, such as afb1-5 and afb3-4, are not null (see Figure S3), it is possible that other auxin receptors might be responsible for the regulation of flowering time. We also examined the flowering phenotype of a variety of arf mutants, and found that the arf7-1 arf19-1 double mutant flowered later than the WT under SDs in terms of days to flowering (see Table S1). With regard to the number of rosette leaves at flowering, the arf7-1 arf19-1 mutant plants did not show a pronounced late flowering phenotype (see Table S2). This discrepancy might be correlated with functional redundancy by other ARFs. This possibility can be tested by the genetic crossing of arf7 arf19 mutant with other arf mutants, and by a further flowering-time analysis of the resulting triple, quadruple, and quintuple mutants in future studies.
In summary, we demonstrate here that IAA7/AXR2 acts to inhibit flowering time under SDs, at least in part, by negatively regulating the expression of GA20ox1 and GA20ox2 genes. Our results suggest that auxin might be implicated in the regulation of flowering time under SDs. However, major questions remain to be answered in future studies. For example, are there any ARFs that are negatively regulated by IAA7/AXR2 in regulating the flowering time? Do auxin receptors that act dependently or independently of the TIR1/AFBs proteins to regulate floral initiation exist? Is the exogenous application of auxin able to promote flowering under SDs, and can auxin-deficient mutants show a later flowering phenotype under SDs?
Materials and Methods
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- Materials and Methods
- Supporting Information
Plant materials and growth conditions
The Arabidopsis mutants used for this report are from the Columbia ecotype line. axr2-1 (CS3077), spy-1 (CS6266), spy-3 (CS6268), arf1-3 (CS24599), arf3-2 (CS24604), arf4-1 (SALK_023804c), arf6-1 (CS24606), arf7-1 (CS24607), arf8-2 (CS24608), arf9-1 (CS24609), arf10-1 (CS24611), arf11 (CS24612), arf12-1 (CS24613), arf13-1 (CS24614), arf14 (SALK_049581c), arf15-1 (CS24615), arf16-1 (CS24616), arf17-2 (SALK_138426), arf19-1 (CS24617), arf20-1 (CS24619), arf21-1 (CS24621), arf22-1 (CS24623), arf6-1 arf8-2 (CS24632), arf7-1 arf19-1 (CS24629), tir1-1 (CS3798), afb1-5 (SALK_144884), afb2-3 (SALK_137151), and afb3-4 (SALK_068787) were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA) (Alonso et al. 2003). Seeds were sterilized with 20% bleach containing 0.1% Tween-20 for 15 min, and washed with four changes of sterile water before being sown on MS basal medium (Sigma, St Louis, MO, USA). Plates were kept in the dark at 4 °C for 3–5 d before germination in either LD light conditions (16 h light/8 h dark) or SD light conditions (9 h light/15 h dark), with a fluence rate of 120 μmol m−2 s−1 of white light (cool-white fluorescent lamps). Seven-day-old seedlings were transplanted into soil.
Construction of double, triple, and quadruple mutants
The single mutants axr2-1, spy-1, spy-3, arf3-2, arf4-1, tir1-1, afb1-5, afb2-3, and afb3-4 were used to construct the axr2-1 spy-1, axr2-1 spy-3, arf3-2 arf4-1, tir1-1 afb2-3, and afb1-5 afb3-4 double, tir1-1 afb2-3 afb3-4 triple, and tir1-1 afb1-5 afb2-3 afb3-4 quadruple mutants. Mutations of AXR2, SPY, and TIR1 were verified by DNA sequencing, and those of ARF3, ARF4, AFB1, AFB2, and AFB3 were characterized by PCR. To select the tir1-1 afb2-3 afb3-4 triple and tir1-1 afb1-5 afb2-3 afb3-4 quadruple mutants, F2 seedlings were screened in the dark, and seedlings with extremely short hypocotyls were chosen. The homozygous tir1-1 afb2-3 afb3-4 triple and tir1-1 afb1-5 afb2-3 afb3-4 quadruple mutants were further confirmed by PCR and DNA sequencing, and phenotypic analysis in F3 and F4 generations. Primers used are shown in Table S3.
Construction of plant-expression cassettes, plant transformation, and characterization of transgenic plants
Polymerase chain reaction-amplified fragments encoding 3xHA were cloned into SpeI and SacI sites of pBluescript SK (pBS). The iaaL fragment was synthesized by PCR using the pYL608 vector harboring the iaaL gene (Yang et al. 1997), and cloned into the BamHI and SpeI sites of pBS-3xHA, resulting in pBS-iaaL-3xHA. The PCR-amplified full-length SOC1 fragment was cloned into BamHI and SpeI sites of pBS-3xHA, resulting in pBS-SOC1-3xHA. The PCR amplified 6xMYC fragment was cloned into XhoI and EcoRI sites of pBS. The PCR-amplified, full-length LFY fragment was cloned into EcoRI and SacI sites of pBS-6xMYC, resulting in pBS-6xMYC-LFY. All of the constructs used were confirmed by DNA sequencing. Fragments encoding iaaL-3xHA and SOC1-3xHA were excised by digestion with BamHI and SacI and cloned into pHB (Mao et al. 2005) to generate pHB-iaaL-3xHA and pHB-SOC1-3xHA, respectively. Fragments encoding 6XMYC-LFY were excised by digestion with XhoI and SacI, and cloned into the plant expression vector pKYL71 (Schardl et al. 1987) to generate pKYL71-6xMYC-LFY. All constructs were introduced into Agrobacterium tumefaciens strain GV3101. The floral dipping method was used to create transgenic plants (Bechtold et al. 1993). Transgenic seeds were screened on MS plates containing either 100 mg/mL kanamycin or 50 mg/mL hygromycin. At least 30 independent T1 lines for each construct were generated. Homozygous T4 seeds of 3 representative lines for pHB-iaaL-3xHA and pHB-SOC1-3xHA constructs were routinely used for the phenotypic analyses. Primers used are shown in Table S3.
Seedlings were germinated on MS medium and then transferred to soil and grown in LDs (16 h light/8 h dark) or SDs (9 h light/15 h dark), illuminated by white light at a fluence rate of 120 μmol m−2 s−1. Flowering time was measured by scoring the number of days from germination to flowering and/or the number of rosette leaves. Plants were checked for flower buds every day. At least 30 plants were analyzed under LDs; 20 plants were analyzed under SDs.
Beginning 17 d after planting under SDs, GA-treated plants were sprayed generously once a week with 0.1 mM GA3 (Sigma, USA) and 0.02% (v/v) Tween-20. Control plants were sprayed with a solution containing only the Tween-20 and 0.1% (v/v) dimethylformamide (the solvent for the GA3 stock solution).
Western blot analysis
Total protein was extracted from 8-d-old seedlings. Proteins were fractionated on a 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis mini-gel and blotted on to a nitrocellulose filter. The anti-CCT1 antibody was prepared against Arabidopsis CRY1, as previously described (Sang et al. 2005). The anti-HA antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein detection was conducted using the ECL PLUS kit (Amersham Pharmacia, Piscataway, NJ, USA).
Reverse transcription-polymerase chain reaction and quantitative real-time PCR
Seedlings grown under SDs for approximately 8 d were collected (Figures 5, 6, and Figure S3). Apical meristem/young leaves of soil-grown plants were collected at the time when WT plants (Figure 4A, B) or GA-treated WT plants (Figure 4C, D) flowered. The extractions of total RNA, RT-PCR, and qRT PCR were performed as described previously (Liu et al. 2008; Wang et al. 2010a, b; Wu and Yang 2010), with minor modifications as follows. SYBR green was used as a dye for the quantification of double-stranded DNA. Primers used are shown in Table S3. All experiments were performed with three independent biological replicates and three technical repetitions.
(Co-Editor: Li-Jia Qu)
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- Materials and Methods
- Supporting Information
We thank members of the H.-Q.Y.'s group for discussion and comments on the manuscript. Some stocks were obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA). This work was supported by the National Natural Science Foundation of China (90917014 to H.-Q.Y.), the Ministry of Science and Technology of China (2006AA10A102), the National Special Grant for Transgenic Crops (2009ZX08009–081B to H.-Q.Y.), the Science and Technology Commission of Shanghai Municipality (10XD1402300 to H.-Q.Y.), and the Shanghai Leading Academic Discipline Project (B209).
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- Materials and Methods
- Supporting Information
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- Supporting Information
Figure S1.SPINDLY genetically acts downstream of IAA7 to regulate flowering time. (A) Representative (60-d-old) wild-type, axr2-1, spy-1, spy-1 axr2-1, spy-3, and spy-3 axr2-1 mutant plants grown under short days. Bars = 1 cm. (B) Representative (25-d-old) wild-type, axr2-1, spy-1, spy-1 axr2-1, spy-3, and spy-3 axr2-1 mutant plants grown under long days. Bars = 1 cm. (C) Flowering time expressed as time to flower of wild-type, axr2-1, spy-1, spy-1 axr2-1, spy-3, and spy-3 axr2-1 grown under short days. n ≥ 20; error bars indicate standard deviation. (D) Flowering time expressed as time to flower of wild-type, axr2-1, spy-1, spy-1 axr2-1, spy-3, and spy-3 axr2-1 grown under long days. n ≥ 30; error bars indicate standard deviation.
Figure S2. Overexpression of LEAFY in axr2-1 mutant rescues the late flowering phenotype. (A) Representative (90-d-old) 35Spro–NPTII and 35Spro–NPTII/axr2-1 plants grown under short days. Transgenic 35Spro–NPTII lines flower earlier than 35Spro–NPTII/axr2-1 plants. Bars = 1 cm. (B) Representative (90-d-old) 35Spro–LFY and 35Spro–LFY/axr2-1 plants grown under short days. Transgenic 35Spro–LFY lines and 35Spro–LFY/axr2-1 lines flower early. Bars = 1 cm. (C) Flowering time expressed as time to flower of 35Spro–NPTII, 35Spro–NPTII/axr2-1, 35Spro–LFY, and 35Spro–LFY/axr2-1 plants grown under short days. n ≥ 12; error bars indicate standard deviation.
Figure S3. Characterization of the tir1-1, afb1-5, afb2-3, and afb3-4 mutants. (A) Gene structure showing exons (filled boxes) and introns (black lines) in TIR1, AFB1, AFB2, or AFB3. Position of T-DNA insertions in afb1-5, afb2-3, and afb3-4 alleles is indicated with triangles. Asterisks denote the position of the point mutation tir1-1. (B–E) Reverse transcription–polymerase chain reactions (PCR) were performed using RNA isolated from 8-d-old, short day-grown wild-type and mutant seedlings. Arrows denote positions of primer pairs used in each PCR. Tubulin 2 was used as an internal loading control.
Figure S4. Flowering-time phenotype analysis of the single, double, triple, and quadruple mutants of tir1-1, afb1-5, afb2-3, and afb3-4. (A) Schematic diagrams displaying T-DNA insertions in the AFB1, AFB2, and AFB3 loci of the afb1-5 (SALK_144884), afb2-3 (SALK _137151), afb3-4 (SALK_068787) mutants, respectively. lp, rp, and P1 (Lba1) are the primers used for PCR-genotyping. Triangles indicate T-DNA insertion sites. (B) Polymerase chain reactions analysis of T-DNA insertions in the AFB1, AFB2, and AFB3 loci in tir1-1 afb2-3 double mutants, tir1-1 afb2-3 afb3-4 triple mutants, and tir1-1 afb1-5 afb2-3 afb3-4 quadruple mutants, respectively. (C) Representative (60-d-old) wild-type, tir1-1, tir1-1 afb2-3, tir1-1 afb2-3 afb3-4, and tir1-1 afb1-5 afb2-3 afb3-4 plants grown under short days. Bars = 1 cm. (D) Representative (25-d-old) wild-type, tir1-1, tir1-1 afb2-3, tir1-1 afb2-3 afb3-4, and tir1-1 afb1-5 afb2-3 afb3-4 plants grown under long days. Bars = 1 cm. (E) Flowering time expressed as time to flower of wild-type, tir1-1, tir1-1 afb2-3, tir1-1 afb2-3 afb3-4, and tir1-1 afb1-5 afb2-3 afb3-4 plants grown under short days. n ≥ 20; error bars indicate standard deviation. (F) Flowering time expressed as time to flower of wild-type, tir1-1, tir1-1 afb2-3, tir1-1 afb2-3 afb3-4, and tir1-1 afb1-5 afb2-3 afb3-4 plants grown under long days. n ≥ 30; error bars indicate standard deviation.
Table S1. Flowering time of arf mutants under short days and long days.
Table S2. Flowering time of plants under short days and long days.
Table S3. Primers for mutant genotyping, plasmid construction, reverse transcription–polymerase chain reaction, and quantitative real-time polymerase chain reaction.
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