PIF4–Mediated Activation of Expression Integrates Temperature into the Auxin Pathway in Regulating Hypocotyl Growth
Higher plants adapt their growth to high temperature by a dramatic change in plant architecture. It has been shown that the transcriptional regulator phytochrome-interacting factor 4 (PIF4) and the phytohormone auxin are involved in the regulation of high temperature–induced hypocotyl elongation in Arabidopsis. Here we report that PIF4 regulates high temperature–induced hypocotyl elongation through direct activation of the auxin biosynthetic gene YUCCA8 (YUC8). We show that high temperature co-upregulates the transcript abundance of PIF4 and YUC8. PIF4–dependency of high temperature–mediated induction of YUC8 expression as well as auxin biosynthesis, together with the finding that overexpression of PIF4 leads to increased expression of YUC8 and elevated free IAA levels in planta, suggests a possibility that PIF4 directly activates YUC8 expression. Indeed, gel shift and chromatin immunoprecipitation experiments demonstrate that PIF4 associates with the G-box–containing promoter region of YUC8. Transient expression assay in Nicotiana benthamiana leaves support that PIF4 directly activates YUC8 expression in vivo. Significantly, we show that the yuc8 mutation can largely suppress the long-hypocotyl phenotype of PIF4–overexpression plants and also can reduce high temperature–induced hypocotyl elongation. Genetic analyses reveal that the shy2-2 mutation, which harbors a stabilized mutant form of the IAA3 protein and therefore is defective in high temperature–induced hypocotyl elongation, largely suppresses the long-hypocotyl phenotype of PIF4–overexpression plants. Taken together, our results illuminate a molecular framework by which the PIF4 transcriptional regulator integrates its action into the auxin pathway through activating the expression of specific auxin biosynthetic gene. These studies advance our understanding on the molecular mechanism underlying high temperature–induced adaptation in plant architecture.
Published in the journal:
. PLoS Genet 8(3): e32767. doi:10.1371/journal.pgen.1002594
Category:
Research Article
doi:
https://doi.org/10.1371/journal.pgen.1002594
Summary
Higher plants adapt their growth to high temperature by a dramatic change in plant architecture. It has been shown that the transcriptional regulator phytochrome-interacting factor 4 (PIF4) and the phytohormone auxin are involved in the regulation of high temperature–induced hypocotyl elongation in Arabidopsis. Here we report that PIF4 regulates high temperature–induced hypocotyl elongation through direct activation of the auxin biosynthetic gene YUCCA8 (YUC8). We show that high temperature co-upregulates the transcript abundance of PIF4 and YUC8. PIF4–dependency of high temperature–mediated induction of YUC8 expression as well as auxin biosynthesis, together with the finding that overexpression of PIF4 leads to increased expression of YUC8 and elevated free IAA levels in planta, suggests a possibility that PIF4 directly activates YUC8 expression. Indeed, gel shift and chromatin immunoprecipitation experiments demonstrate that PIF4 associates with the G-box–containing promoter region of YUC8. Transient expression assay in Nicotiana benthamiana leaves support that PIF4 directly activates YUC8 expression in vivo. Significantly, we show that the yuc8 mutation can largely suppress the long-hypocotyl phenotype of PIF4–overexpression plants and also can reduce high temperature–induced hypocotyl elongation. Genetic analyses reveal that the shy2-2 mutation, which harbors a stabilized mutant form of the IAA3 protein and therefore is defective in high temperature–induced hypocotyl elongation, largely suppresses the long-hypocotyl phenotype of PIF4–overexpression plants. Taken together, our results illuminate a molecular framework by which the PIF4 transcriptional regulator integrates its action into the auxin pathway through activating the expression of specific auxin biosynthetic gene. These studies advance our understanding on the molecular mechanism underlying high temperature–induced adaptation in plant architecture.
Introduction
Higher plants continually sense environmental conditions to adapt their growth and development. To a large extent, this is achieved through integrating environmental cues into the growth-regulating hormonal pathways. Exposure of Arabidopsis thaliana plants to high temperature (29°C) results in dramatic plant architecture changes including rapid hypocotyl elongation, leaf hyponasty, and early flowering [1]–[4]. High temperature-induced hypocotyl elongation of Arabidopsis plants provides an ideal model system to investigate the regulatory mechanisms underlying adaptive growth of plants to their ever-changing environments. Among the endogenous cues involved in the regulation of high temperature-induced hypocotyl elongation is the plant hormone auxin [3]. An early observation revealed a correlation between high temperature-induced hypocotyl elongation and high temperature-induced elevation of endogenous free indole-3-acetic acid (IAA) levels [3]. Genetic analyses found that high temperature-induced hypocotyl elongation is sharply reduced in Arabidopsis mutants defective in auxin biosynthesis, transport or signaling [3]. Together, these data attribute an essential role of the auxin pathway in mediating high temperature-induced hypocotyl elongation.
It is long-recognized that auxin has profound effects on plant growth and development. A combination of physiological, biochemical, pharmacological and molecular genetic studies provide an ever-growing body of insights on our understanding of the auxin biosynthesis pathway [5], [6]. It is generally believed that, IAA, the main auxin in higher plants, can be synthesized from tryptophan (Trp)-dependent and -independent pathways [5]. Among the best-characterized enzymes involved in the Trp-dependent auxin biosynthetic pathway are the YUCCA (YUC) family of flavin-containing monooxygenases [5], [7]–[9] and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR) family of aminotransferases [5], [10], [11]. A wealth of genetic evidence indicated that, while inactivating members of the YUC family genes causes dramatic developmental defects [8], [9], overexpression of the YUC family genes leads to auxin overproduction and long hypocotyl phenotype in Arabidopsis [7]. Although mutation of TAA1 or its close homologs (TAR genes) leads to developmental defects similar to those of the yuc mutants [10], [11], overexpression of TAA1/TAR does not cause obvious developmental phenotype, suggesting that TAA1/TAR probably do not catalyze a rate-limiting step in IAA biosynthesis [10], [11]. Interestingly, recent studies provide evidence that TAA1/TARs and YUCs may act in a common linear biosynthetic pathway for auxin production [6], [12], [13].
In addition to auxin, a family of phytochrome-interacting factors (PIFs), which encode basic helix-loop-helix (bHLH) transcription factors, have been shown to be central integrators of versatile environmental and hormonal signals during plant adaptive growth [14], [15]. Among the PIF family of transcriptional regulators, a selective function of PIF4 in high temperature-induced hypocotyl elongation has recently been reported [1], [16]. These studies revealed that high temperature induced a rapid elevation of PIF4 transcript levels and that the pif4 mutant largely lost the robust enhancement of hypocotyl elongation induced by high temperature [1].
In the context that both the transcription factor PIF4 and the phytohormone auxin are required for high temperature-induced hypocotyl elongation, a fascinating hypothesis is that PIF4 may directly link the auxin pathway in regulating plant adaptation growth to high temperature. We provide here evidence that, in response to high temperature, PIF4 directly activates YUC8 expression and thus elevates endogenous free IAA levels. We also show that the SHY2/IAA3 protein is a downstream component of the PIF4-auxin signaling pathway in regulating high temperature-induced hypocotyl elongation. Our results exemplify how a transcriptional regulator integrates environmental cues with endogenous hormonal signaling to mediate specialized developmental changes in regulating plant adaptive growth.
Results
Loss of PIF4 Function Impairs High Temperature–Induced Elevation of YUC8 Transcripts and Endogenous Free IAA Levels
It has been shown that high temperature activates the expression of the transcription factor PIF4 [1], and elevates endogenous free IAA levels [3] in Arabidopsis. To explore the possible molecular linkage between PIF4 and the auxin pathway in regulating high temperature-mediated adaptation growth, we examined high temperature-induced expression of PIF4 and the YUCCA (YUC) family of auxin biosynthetic genes [5]. Consistent with previous reports [1], , when wild type (WT) seedlings grown at 22°C for 6 days were transferred to 29°C in continuous light over a 24 h time course, PIF4 transcript abundance was transiently elevated to a peak level at 3 h after transfer (Figure 1A). Correlating with an increased expression of PIF4, high temperature also markedly increased transcript abundance of YUC8 with a peak at 3 h in WT seedlings (Figure 1B). Closer observation with a narrower range of time points revealed that high temperature-mediated induction of YUC8 expression occurred generally later than that of PIF4 (Figure S1). Parallel experiments indicated that high temperature did not upregulate the expression of other YUCCA family genes tested (Figure S2). We then compared high temperature-induced YUC8 expression between WT and the pif4 mutant, which has been shown to be defective in high temperature-induced adaptations in plant architecture (Figure 1B). As shown in Figure 1B, the basal expression levels of YUC8 were already low in pif4 seedlings and, significantly, high temperature-induced upregulation of YUC8 expression was largely abolished in this mutant, indicating that the function of PIF4 is important for the basal- and high temperature-induced expression of YUC8.
The pif4 mutation impairs high temperature-induced upregulation of YUC8 expression suggests that this mutation may also affect high temperature-induced elevation of free IAA levels. To test this, we compared high temperature-induced elevation of free IAA levels in WT and pif4 seedlings. For these experiments, we grew seedlings at 22°C or 29°C in continuous light for 6 days and collected hypocotyls for IAA measurement. Consistent with a previous report [3], high temperature increased free IAA levels of WT seedlings by around 50% (Figure 1C). As expected, high temperature-induced elevation of free IAA levels was abolished in the pif4 mutant (Figure 1C), indicating that PIF4 is also required for high temperature-induced elevation of auxin biosynthesis. Together, these results suggest that PIF4 and YUC8 may function in linking temperature and auxin pathway in regulating hypocotyl elongation.
Overexpression of PIF4 Upregulates YUC8 Expression and Leads to Elevated Endogenous Free IAA Levels
As a first step to test the possibility that PIF4 may directly regulate YUC8 expression during high temperature-induced adaptation growth, we examined YUC8 expression in transgenic plants overexpressing PIF4 (35S-PIF4). Like the reported yucca mutants which contain increased endogenous auxin levels [7], 35S-PIF4 plants show a long hypocotyl phenotype that resembles high temperature-grown WT seedlings (Figure S3). As revealed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays, the expression of YUC8 (Figure 2A), but not that of TAA1 (Figure S4), was substantially increased in 35S-PIF4 seedlings as compared to WT. We also generated PIF4-overexpression plants (pMDC7:PIF4) using the chemical inducible vector pMDC7 [17]. In the presence of the chemical inducer estradiol, pMDC7:PIF4 seedlings show increased expression of PIF4 (Figure 2B) and display a long hypocotyl phenotype like 35S-PIF4 seedlings (Figure S5). As expected, YUC8 expression was considerably elevated following estradiol induction (Figure 2C). Consistently, measurement of auxin revealed that the free IAA levels in 35S-PIF4 plants were increased by 50% as compared to those in WT plants (Figure 2D). In line with increased free IAA levels in 35S-PIF4 plants, the expression of the auxin responsive DR5:GUS, a widely used reporter of auxin response, was clearly enhanced in the basal region of 35S-PIF4 hypocotyls (Figure S6). These data together indicate that overexpression of PIF4 leads to increased expression of the auxin biosynthetic gene YUC8 and, as a result, elevated endogenous free IAA levels in planta.
PIF4 Directly Binds to the Promoter Region of YUC8
Three lines of evidence support a scenario that the PIF4 transcription factor may directly regulate YUC8 expression during high temperature-induced adaptation growth. First, underlying high temperature-induced hypocotyl elongation, high temperature upregulates the expression of PIF4 in a similar fasion to that of YUC8. Second, high temperature-induced upregulation of YUC8 expression requires the function of PIF4. Third, overexpression of PIF4 leads to increased expression of YUC8 and elevated free IAA levels in planta. Given that PIF4 specifically binds to a core DNA G-box motif (CACGTG) of its target gene promoters [18], we searched for the presence of G-box motifs in the promoter regions of the 11 YUC family genes present in the Arabidopsis genome. As shown in Figure 3A, G-box motifs were found not only in the promoter of YUC8, whose expression was significantly induced by high temperature (Figure 1), but also in the promoters of YUC5, YUC9 and YUC10, whose expression was not or slightly induced by high temperature (Figure S2). To test the idea that PIF4 may actually bind to the G-box-containing regions of these YUC genes, we performed chromatin immuno-precipitaiton (ChIP) assays using a previously reported transgenic line expressing a fusion of PIF4 to the haemagglutinin (HA) antigen (PIF4-HA) [19] and anti-HA antibody (Abcam). PCR amplification of the promoter regions of the four YUC genes showed that PIF4-HA specifically bound to the G-box-containing promoter region of YUC8, but not to the G-box-containing promoter regions of YUC5, YUC9 and YUC10 (Figure 3B). These results suggest that PIF4 associates with the G-box DNA motifs in the promoter region of YUC8 in vivo. Further evidence supporting this conclusion came from electrophoretic mobility-shift assays (EMSA) using PIF4 protein expressed in vitro. As shown in Figure 3C, PIF4 bound to the G-box-containing DNA fragments present in the promoter region of YUC8 and, this binding could be effectively competed by the addition of excess amount of unlabeled G-box-containing DNA probes (Figure 3C). As a control, we showed that DNA probes containing a mutated G-box motif (CACGGG) failed to compete the binding of PIF4 to the G-box-containing DNA fragments (Figure 3C). Together, these results support that the PIF4 transcription factor regulates YUC8 expression by directly binding to its promoter region.
PIF4 Activates YUC8 Expression in the Transient Expression Assay
Next, using the well-established transient expression assay of Nicotiana benthamiana leaves, we verified the activation effect of PIF4 on the expression of a reporter containing the YUC8 promoter fused with the firefly luciferase (LUC) gene. When the pYUC8:LUC reporter was infiltrated into N. benthamiana, the LUC activity could be detected at lower level (Figure 4A, B). Coexpression of pYUC8:LUC with the 35S:PIF4 construct led to an obvious induction in luminescence intensity (Figure 4A, 4B), suggesting that ectopic expression of PIF4 can activate pYUC8:LUC expression in this transient expression assay. In a parallel experiment, pYUC8(mut):LUC, in which the two G-boxes of the YUC8 promoter were deleted and fused with LUC, togehter with 35S:PIF4 were co-infiltrated into N. benthamiana leaves. As shown in Figure 4, the activation effect of PIF4 on pYUC8(mut):LUC expression was abolished. Together, our transient expression assays in N. benthamiana leaves confirmed that PIF4 directly activates YUC8 expression in vivo.
The YUC8 Gene Is Required for PIF4–Mediated Hypocotyl Elongation
To determine the genetic relationship between PIF4 and YUC8, we identified a yuc8 mutant (SALK_096110) which harbors a T-DNA insertion that markedly reduced the expression levels of the YUC8 gene (Figure S7). We show that the yuc8 mutant is defective in high temperature-induced hypocotyl growth (Figure 5A). We then introduced the above-described 35S-PIF4 construct into the genetic background of the yuc8 mutant through genetic crossing. As shown in Figure 5B, the yuc8 mutation substantially suppressed the long-hypocotyl phenotype of the 35S-PIF4 plants, supporting that YUC8 acts genetically downstream of PIF4 in regulating high temperature-induced hypocotyl elongation.
SHY2/IAA3 Is an Important Component of the PIF4–Auxin Pathway in Regulating High Temperature–Induced Hypocotyl Elongation
Several elegant observations have demonstrated the involvement of PIF4 and auxin in regulating adaptive growth of plants to high temperature [1], [16]. Our data presented here further revealed that, through directly activating of the YUC8 expression, PIF4 integrates its action into the auxin pathway in regulating high temperature-mediated hypocotyl elongation. To further identify auxin signaling components involved in this process, we employed a genetic approach to search for auxin-related mutations that can suppress the long-hypocotyl phenotype of the 35S-PIF4 plants. It has been shown that the shy2-2 mutant, which harbors a stabilized mutant form of the SHY2/IAA3 protein, displays a short hypocotyl phenotype [20], suggesting a role of SHY2/IAA3 in regulating auxin-mediated hypocotyl growth. We showed that shy2-2 seedlings are defective in high temperature-induced hypocotyl growth (Figure S3). Importantly, like the auxin signaling mutant axr1-12 (Figure S8), shy2-2 genetically suppressed the long-hypocotyl phenotype of 35S-PIF4 (Figure 6). In contrast, other gain-of-function mutations in different IAA proteins [21]–[24], including slr-1 (contains a gain-of-function mutation in IAA14), axr2-1 (contains a gain-of-function mutation in IAA7), axr5-1 (contains a gain-of-function mutation in IAA1) and iaa28-1, did not affect hypocotyl elongation in response to high temperature and failed to suppress the long-hypocotyl phenotype of 35S-PIF4 seedlings (Figure S9). These results demonstrate that the auxin signaling repressor SHY2/IAA3 is selectively involved in high temperature-induced hypocotyl growth.
Discussion
As sessile organisms, plants have evolved remarkable ability to adapt their development to the ever-changing environmental conditions. Exposure of plants to high temperature results in dramatic changes in plant architecture, including elongation responses and leaf hyponasty. High temperatures can also considerably reduce plant biomass, raising concerns over future crop productivity and food security. Therefore, the modulation of plant architecture by high temperature is a subject of considerable agricultural significance, particularly with regard to global climate change. An ever-growing body of evidence in Arabidopsis has implicated that high temperature-induced plant architecture remodeling relies on the interplays between multiple external and internal cues including light, circadian clock, auxin, gibberellin and others [25], [26]. Particularly, recent studies reveal that a group of bHLH transcription factors play a central role in modulating developmental responses to both light and temperature [1], [14], [16], [27]–[29].
PIF4 Is an Integrator between High Temperature and Auxin Pathway in Regulating Adaptive Hypocotyl Growth
In this study, we discovered that, as a molecular integrator, the PIF4 transcription factor links high temperature to the auxin pathway in regulating high temperature-induced hypocotyl elongation. Several lines of evidence support this finding: First, underlying the long-standing observation that high temperature induces a dramatic elongation of the hypocotyl, we showed that high temperature triggers an elevation of the transcript abundance of both PIF4 and YUC8 (Figure 1). Second, high temperature-induced upregulation of YUC8 expression largely depends on the function of PIF4 (Figure 1). Third, overexpression of PIF4 leads to increased expression of YUC8 and elevated endogenous free IAA levels (Figure 2). Fourth, as revealed by ChIP and EMSA assays, PIF4 specifically binds to a core DNA G-box motif (CACGTG) present in the promoter of the YUC8 gene (Figure 3). Fifth, transactivation assays in N. benthamiana leaves support that PIF4 stimulates the activity of the YUC8 promoter fused with a reporter (Figure 4). Finally, the yuc8 mutation, which is defective in high temperature-induced hypocotyl elongation, is able to partially suppress the long-hypocotyl phenotype of the 35S-PIF4 plants (Figure 5). Together, these data support that, PIF4 selectively activates the expression of the auxin biosynthetic gene YUC8, thus integrates high temperature to the auxin pathway in regulating adaptive hypocotyl growth.
It is worthy of note that the yuc8 mutant still retains some response to high temperature in hypocotyl elongation and that this mutation fails to completely suppress the long-hypocotyl phenotype of 35S-PIF4 plants (Figure 5). A plausible explanation for this is that the yuc8 mutant used in this study shows reduced, but not loss of, YUC8 expression (Figure S7). Alternatively, we could not rule out the possibility that PIF4 may activate auxin biosynthetic genes other than YUC8, which act weakly in PIF4-mediated hypocotyl growth in response to high temperature. A very recent report hints that the PIF4 transcription factor could target TAA1 [29], which acts genetically upstream of the YUC family genes in IAA production [12], [13]. Considering that overexpression of TAA1 does not lead to any obvious developmental phenotype [11], [12] and that TAA1 and YUCs act in a common linear biosynthetic pathway for auxin production [6], [12], [13], it is reasonable to propose that TAA1 acts together with other auxin biosynthesis genes such as YUC8 to mediate high temperature-induced and PIF4-mediated hypocotyl elongation. However, our gene expression analyses reveal that overexpression of PIF4 alone fails to elevate TAA1 transcription (Figure S4).
PIF4-mediated activation of YUC8 expression in response to high temperature exemplifies a mechanism by which environmental cues manipulate auxin, the key endogenous modulator of plant architecture. Another known physiological process in which both PIF4 and auxin are involved is shade avoidance syndrome (SAS), plant adaptive growth responses to the light signal [14], [30], [31]. PIF4 is therefore emerging as a molecular “hub” to integrate both temperature and light signals to regulate plant architecture remodeling [14]. Accumulating evidence reveals that, unlike shade avoidance, where PIF4 acts redundantly with its homolog, PIF5, to regulate elongation growth, PIF4 appears to perform a dominant role in driving high temperature-induced adaptive growth [1], [14], [16], [32]–[34]. These studies suggest that, PIF4, and possibly other PIF family members, have specialized and overlapping functions in regulating plant adaptive growth to different environmental stimuli.
SHY2/IAA3 Is Specifically Involved in PIF4–Mediated Hypocotyl Elongation in Response to High Temperature
Our results support a scenario in which the auxin pathway acts downstream of the PIF4 transcriptional regulator in regulating high temperature-induced hypocotyl elongation. Supporting evidence for this hypothesis came from our genetic analysis showing that the axr1-12 mutation, which contains a mutation in a subunit of the heterodimeric RUB-E1 enzyme required for auxin signaling [35], completely suppressed the long-hypocotyl phenotype of 35S-PIF4 seedlings (Figure S8). Based on our current knowledge of the auxin signaling pathway, auxin mediates the expression of auxin responsive genes through the inactivation of AUX/IAA transcriptional repressors that negatively control the activity of AUXIN REPONSE FACTOR (ARF) transcription factors [36]. In the context that many gain-of-function aux/iaa mutations are associated with reduced response to exogenous auxin, but developmental defects among these mutants are frequently more specific [36], it is reasonable to speculate that specific Aux/IAA-ARF pair(s) may function in the PIF4-auxin pathway to mediate the specialized hypocotyl elongation process triggered by high temperature. In our genetic efforts to identify new components involved in the PIF4-auxin pathway in regulating high temperature-mediated hypocotyl elongation, we determined that SHY2/IAA3, but not other IAA proteins tested, has a specialized function in mediating high temperature-induced hypocotyl elongation. It is of interest in future studies to identify the ARF transcription factor(s) interacting with SHY2/IAA3 in regulating high temperature-induced hypocotyl elongation.
Materials and Methods
Plant Materials and Growth Conditions
Arabidopsis thaliana ecotypes Columbia (Col-0), Ler and WS were used as wild types. The pif4 mutant used in this study was the reported null allele pif4-2 [1]. Other plant materials used in this study were previously described: DR5:GUS [37], 35S-PIF4 [19], 35S:PIF4-HA [19], yucca [7], axr1-12 [38], shy2-2 [2019], slr-1 [21], axr2-1 [22], axr5-1 [23] and iaa28-1 [24]. yuc8 (SALK_096110) was identified from the SIGnAL T-DNA collection [39].
All molecular manipulations were performed according to standard methods [40]. The PIF4 coding fragment was amplified by PCR and cloned into the AscI/PacI sites of the binary vector pMDC7 [17], resulting in a chemical-inducible PIF4 expression construct. The construct was then transformed into Agrobacterium tumefaciens strain GV3101 (pMP90), which was used for transformation of Arabidopsis plants by vacuum infiltration [41].
Seeds were surface-sterilized for 15 min in 10% bleach, washed four times with sterile water, and plated on half-strength Murashige and Skoog (MS) medium. Plants were stratified at 4°C for 2 d in darkness and then transferred to a phytotrone set at 22°C with a 16-h light/8-h dark photoperiod or in continuous light for specific experiments. For high temperature treatment, plants were directly grown at 29°C in continuous light or young seedlings were transferred to 29°C in continuous light for different times.
Gene Expression Analysis
For qRT-PCR analysis, seedling were harvested and frozen in liquid nitrogen for RNA extraction. RNA extraction and qRT-PCR analysis were performed as previously described [37]. Primers used to quantify gene expression levels are listed in Table S1. The GUS activity assays were performed as previously described [37].
ChIP–PCR Assay
One gram of 6-d-old seedlings of 35S:PIF4-HA transgenic plants [19] and the anti-HA antibody (Abcam) were used in ChIP experiments. Chromatin immunoprecipitation (ChIP) assays were performed as previously described [42]. The enrichment of DNA fragments was determined by semi-quantitative PCR analysis. Three independent biological repeats were performed.
DNA Gel-Shift Assay
PIF4 and Luciferase (Luc) were synthesized by using the Rabbit Reticulocyte TNT system (Promega) [18], [43]. The 60-bp YUC8 promoter probes containing G-box motifs were synthesized and labeled with biotin at the 3′ end (Invitrogen). Cold competitor probes were generated from dimerized oligos of the YUC8 promoter region containing the wt-G-box (CACGTG) or mut-G-box (CACGGG) motifs, respectively. DNA gel-shift assays were performed as described [18], [43]. Probe sequences are shown in Table S1.
Transactivation of YUC8 Promoter Activity by PIF4 in N. benthamiana Leaves
The transient expression assays were performed in N. benthamiana leaves as previously described [44]. The YUC8 promoter was amplified with the primer pairs 5-CACCATCCGATATGATAACGAT-3 and 5-TGGAAGTTGTATTGGAAA-3 and cloned into pENTR using the pENTR Directional TOPO cloning kit (Invitrogen). To generate YUC8 promoter with mutations, site-directed mutagenesis was used to delete the two G-boxes in the YUC8 promoter (Figure 3) using the TaKaRa MutanBEST kit. Then, the two YUC8 promoter versions were fused with the luciferase reporter gene LUC through the Gateway reactions into the plant binary vector pGWB35 [45] to generate the reporter constructs pYUC8:LUC and pYUC8(mut):LUC. The PIF4 effector construct was the 35S:PIF4. For this construct, the PIF4 coding fragment was amplified by PCR with the primer pairs 5-CACCATGGAACACCAAGGTTGGAG-3 and 5-GTGGTCCAAACGAGAACCGT-3. Five independent determinations were assessed. Error bars represent SD. The experiments were repeated at least five times with similar results.
Free IAA Measurement
For measurement of free IAA levels in wild-type and pif4 mutant hypocotyls in response to high temperature treatment, the hypocotyls of 6-d-old wild-type and pif4 mutant seedlings grown at 22°C and 29°C in continuous light, respectively, were harvested for free IAA measurement. For the wild-type seedlings grown at 29°C, the 2 mm length parts for each hypocotyl (above the junction between hypocotyl and root) were harvested for free IAA measurement. Eight-d-old seedlings of wild-type and 35S-PIF4 grown at 22°C in continuous light were harvested for free IAA measurement. Approximately 200 mg (fresh weight) of tissues were used for IAA extraction and measurement as previously described [46].
Supporting Information
Zdroje
1. KoiniMAAlveyLAllenTTilleyCAHarberdNP 2009 High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol 19 408 413
2. BalasubramanianSSureshkumarSLempeJWeigelD 2006 Potent induction of Arabidopsis thaliana flowering by elevated growth temperature. PLoS Genet 2 e106 doi:10.1371/journal.pgen.0020106
3. GrayWMOstinASandbergGRomanoCPEstelleM 1998 High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis. Proc Natl Acad Sci USA 95 7197 7202
4. KumarSVWiggePA 2010 H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis. Cell 140 136 147
5. ZhaoY 2010 Auxin biosynthesis and its role in plant development. Annu Rev Plant Biol 61 49 64
6. MashiguchiKTanakaKSakaiTSugawaraSKawaideH 2011 The main auxin biosynthesis pathway in Arabidopsis. Proc Natl Acad Sci USA 108 18512 18517
7. ZhaoYChristensenSKFankhauserCCashmanJRCohenJD 2001 A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291 306 309
8. ChengYDaiXZhaoY 2006 Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev 20 1790 1799
9. ChengYDaiXZhaoY 2007 Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis. Plant Cell 19 2430 2439
10. TaoYFerrerJLLjungKPojerFHongF 2008 Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell 133 164 176
11. StepanovaANRobertson-HoytJYunJBenaventeLMXieDY 2008 TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell 133 177 191
12. WonCShenXMashiguchiKZhengZDaiX 2011 Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis. Proc Natl Acad Sci USA 108 18518 18523
13. StepanovaANYunJRoblesLMNovakOHeW 2011 The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis. Plant Cell 23 3961 3973
14. LeivarPQuailPH 2011 PIFs: pivotal components in a cellular signaling hub. Trends Plants Sci 16 19 28
15. LauOSDengXW 2010 Plant hormone signaling lightens up: integrators of light and hormones. Curr Opin Plant Biol 13 571 577
16. StavangJAGallego-BartolomeJGomezMDYoshidaSAsamiT 2009 Hormonal regulation of temperature-induced growth in Arabidopsis. Plant J 60 589 601
17. CurtisMDGrossniklausU 2003 A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133 462 469
18. MoonJZhuLShenHHuqE 2008 PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis. Proc Natl Acad Sci USA 105 9433 9438
19. de LucasMDaviereJMRodriguez-FalconMPontinMIglesias-PedrazJM 2008 A molecular framework for light and gibberellin control of cell elongation. Nature 451 480 484
20. TianQUhlirNJReedJW 2002 Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression. Plant Cell 14 301 319
21. FukakiHTamedaSMasudaHTasakaM 2002 Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J 29 153 168
22. NagpalPWalkerLMYoungJCSonawalaATimpteC 2000 AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol 123 563 574
23. YangXLeeSSoJHDharmasiriSDharmasiriN 2004 The IAA1 protein is encoded by AXR5 and is a substrate of SCF(TIR1). Plant J 40 772 782
24. RoggLELasswellJBartelB 2001 A gain-of-function mutation in IAA28 suppresses lateral root development. Plant Cell 13 465 480
25. PatelDFranklinKA 2009 Temperature-regulation of plant architecture. Plant Signal Behav 4 577 579
26. FranklinKAKnightH 2010 Unravelling plant temperature signalling networks. New Phytol 185 8 10
27. CassonSAFranklinKAGrayJEGriersonCSWhitelamGCHetheringtonAM 2009 Phytochrome B and PIF4 regulate stomatal development in response to light quantity. Curr Biol 19 229 234
28. LucyshynDWiggePA 2009 Plant development: PIF4 integrates diverse environmental signals. Curr Biol 19 R265 266
29. FranklinKALeeSHPatelDKumarSVSpartzAK 2011 PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) regulates auxin biosynthesis at high temperature. Proc Natl Acad Sci USA 108 20231 20235
30. HallidayKJMartinez-GarciaJFJosseEM 2009 Integration of light and auxin signaling. Cold Spring Harb Perspect Biol 1 a001586
31. FranklinKA 2008 Shade avoidance. New Phytol 179 930 944
32. NozueKHarmerSLMaloofJN 2011 Genomic analysis of circadian clock-, light-, and growth-correlated genes reveals PHYTOCHROME-INTERACTING FACTOR5 as a modulator of auxin signaling in Arabidopsis. Plant Physiol 156 357 372
33. LorrainSAllenTDuekPDWhitelamGCFankhauserC 2008 Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J 53 312 323
34. HornitschekPLorrainSZoeteVMichielinOFankhauserC 2009 Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers. EMBO J 28 3893 3902
35. PozoJCTimpteCTanSCallisJEstelleM 1998 The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science 280 1760 1763
36. ChapmanEJEstelleM 2009 Mechanism of auxin-regulated gene expression in plants. Annu Rev Genet 43 265 285
37. SunJXuYYeSJiangHChenQ 2009 Arabidopsis ASA1 is important for jasmonate-mediated regulation of auxin biosynthesis and transport during lateral root formation. Plant Cell 21 1495 1511
38. LincolnCBrittonJHEstelleM 1990 Growth and development of the axr1 mutants of Arabidopsis. Plant Cell 2 1071 1080
39. AlonsoJMStepanovaANLeisseTJKimCJChenH 2003 Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301 653 657
40. SambrookJRussellD 2001 Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
41. BechtoldNPelletierG 1998 In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82 259 266
42. GendrelAVLippmanZMartienssenRColotV 2005 Profiling histone modification patterns in plants using genomic tiling microarrays. Nat Methods 2 213 218
43. HuqEQuailPH 2002 PIF4, a phytochrome-interacting bHLH factor, functions as a negative regulator of phytochrome B signaling in Arabidopsis. EMBO J 21 2441 2450
44. ChenQSunJZhaiQZhouWQiL 2011 The basic helix-loop-helix transcription factor MYC2 directly represses PLETHORA expression during jasmonate-mediated modulation of the root stem cell niche in Arabidopsis. Plant Cell 23 3335 3352
45. NakagawaTKuroseTHinoTTanakaKKawamukaiM 2007 Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104 34 41
46. ZhouWWeiLXuJZhaiQJiangH 2010 Arabidopsis Tyrosylprotein sulfotransferase acts in the auxin/PLETHORA pathway in regulating postembryonic maintenance of the root stem cell niche. Plant Cell 22 3692 3709
Štítky
Genetika Reprodukční medicínaČlánek vyšel v časopise
PLOS Genetics
2012 Číslo 3
- Primární hyperoxalurie – aktuální možnosti diagnostiky a léčby
- Srdeční frekvence embrya může být faktorem užitečným v předpovídání výsledku IVF
- Akutní intermitentní porfyrie
- Vztah užívání alkoholu a mužské fertility
- Šanci na úspěšný průběh těhotenství snižují nevhodné hladiny progesteronu vznikající při umělém oplodnění
Nejčtenější v tomto čísle
- PIF4–Mediated Activation of Expression Integrates Temperature into the Auxin Pathway in Regulating Hypocotyl Growth
- Metabolic Profiling of a Mapping Population Exposes New Insights in the Regulation of Seed Metabolism and Seed, Fruit, and Plant Relations
- A Splice Site Variant in the Bovine Gene Compromises Growth and Regulation of the Inflammatory Response
- Comprehensive Research Synopsis and Systematic Meta-Analyses in Parkinson's Disease Genetics: The PDGene Database