Repression by an auxin/indole acetic acid protein connects auxin signaling with heat shock factor-mediated seed longevity

Raúl Carranco; José Manuel Espinosa; Pilar Prieto-Dapena; Concepción Almoguera; Juan Jordano

doi:10.1073/pnas.1014856107

New Research In

Edited* by Mark Estelle, University of California at San Diego, La Jolla, CA, and approved November 3, 2010 (received for review October 4, 2010)
↵¹R.C., J.M.E., and P.P.-D. contributed equally to this work.

Abstract

The plant hormone auxin regulates growth and development by modulating the stability of auxin/indole acetic acid (Aux/IAA) proteins, which in turn repress auxin response factors (ARFs) transcriptional regulators. In transient assays performed in immature sunflower embryos, we observed that the Aux/IAA protein HaIAA27 represses transcriptional activation by HaHSFA9, a heat shock transcription factor (HSF). We also found that HaIAA27 is stabilized in immature sunflower embryos, where we could show bimolecular fluorescence complementation interaction between native forms of HaIAA27 and HaHSFA9. An auxin-resistant form of HaIAA27 was overexpressed in transgenic tobacco seeds, leading to effects consistent with down-regulation of the ortholog HSFA9 gene, effects not seen with the native HaIAA27 form. Repression of HSFs by HaIAA27 is thus likely alleviated by auxin in maturing seeds. We show that HSFs such as HaHSFA9 are targets of Aux/IAA protein repression. Because HaHSFA9 controls a genetic program involved in seed longevity and embryonic desiccation tolerance, our findings would suggest a mechanism by which these processes can be auxin regulated. Aux/IAA-mediated repression involves transcription factors distinct from ARFs. This finding widens interpretation of auxin responses.

HaHSFA9 and the ortholog factors (HSFA9) are specialized heat shock transcription factors (HSFs) that are expressed only in seeds and perform functions during embryogenesis at normal growth temperature. The heat stress response in plants involves multiple HSFs, but HSFA9 does not have a role in the vegetative response to high temperature (1, 2). In Arabidopsis, transcription of the HSFA9 gene is activated by ABA-insensitive 3 (ABI3), a key regulator controlling late-seed development (2). Target genes of HSFA9 encode different heat shock proteins (HSP) (1–5). Gain of function (3, 4) and loss of function (5) approaches determined that in sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.), HSFA9 activate transcription of specific small heat-shock protein (shsp) genes. Our previous studies (3–5) indicated that HSFA9 factors are involved in the control of a genetic program that regulates seed longevity and embryonic desiccation tolerance. This program includes genes that encode different HSP but not late embryogenesis abundant (LEA) proteins (3–5). To search for additional transcription factors (TFs) involved in the regulation of this process, we used a yeast two-hybrid system to identify embryo TFs that interact with HaHSFA9. Surprisingly, we found that the auxin/indole acetic acid (Aux/IAA) protein HaIAA27 interacts with HaHSFA9.

Aux/IAA are unstable proteins that are further destabilized in response to the major naturally occurring auxin, indole-3-acetic acid (IAA) (6). Aux/IAA proteins act as nuclear-localized transcriptional repressors of auxin response factors (ARFs) (7). In the current model of Aux/IAA function, auxin alleviates repression of ARFs by inducing Aux/IAA degradation in the 26S proteasome (i.e., refs. 8–10 and references therein). Crucial protein–protein interactions in that model have only recently been confirmed. Thus the transport inhibitor response (TIR1) protein, the F-box subunit of the ubiquitin ligase complex SCF^TIR1, is a coreceptor for auxin and interacts with Aux/IAA through amino acids in conserved domain II of Aux/IAA proteins (11–13). Active repression of ARF by at least some Aux/IAA involves protein interaction between transcriptional corepressor TOPLESS (TPL) and amino acids in conserved domain I of Aux/IAAs (14). Finally, the interaction between ARFs and Aux/IAAs occurs in vitro (13–15), or in heterologous systems such as yeast (by two-hybrid) (15–17) or human cells (18). However, confirmation in planta of the interaction between Aux/IAA and ARFs required Aux/IAA protein stabilization by mutation of the domain II amino acids involved in TIR1 binding. In addition, reports for protein–protein interactions involving Aux/IAAs and TFs (or proteins) different from ARFs and TIR1 are very scarce (19, 20). Further, an in planta interaction between an Aux/IAA and TFs distinct from ARF has not been demonstrated.

In this report, we show a direct interaction between HaIAA27 and HaHSFA9 and confirm that this interaction occurs in planta by means of bimolecular fluorescence complementation (BiFC) assays performed within immature zygotic embryos of sunflower. By transient assays performed in these embryos we show that HaIAA27 can repress transcriptional activation of shsp promoters induced by HaHSFA9. In agreement with these findings, seed-specific overexpression in transgenic tobacco plants of an auxin-resistant (domain II mutant) form of HaIAA27, but not that of native HaIAA27, caused effects similar to loss of function of HSFA9 (5). Thus, for example, a reduction in the accumulation of specific sHSPs (cytosolic polypeptides of class CI), encoded by putative target genes of HSFA9 in tobacco, was observed. These findings suggest that repression of HSFA9 by HaIAA27 occurs in planta. In seed immature embryos, HaIAA27 would be stabilized and repress HaHSFA9. In maturing seeds, auxin might destabilize HaIAA27, alleviating that repression; as a consequence, induction of the HaHSFA9 genetic program would be enhanced. Our discovery that not only ARFs but also HSFs are targets of Aux/IAA repression broadens the spectrum and possible explanations of auxin-mediated Aux/IAA protein action. We suggest that Aux/IAA proteins such as HaIAA27 function as repressors of HSF-mediated gene expression program(s) in plant seeds. Our findings support a unique response pathway by which auxin might have effects through an Aux/IAA, but not involving ARF repression.

Results

The same sunflower immature embryo cDNA library used for the isolation of HaHSFA9 (1) was screened by yeast two-hybrid protein interaction cloning, using as bait HaHSFA9. Our screen recovered an Aux/IAA protein, which based on its predicted amino acid sequence and phylogenetic comparisons, we named HaIAA27 (GenBank accession no. FR669188). Thus, among the 29 Aux/IAA proteins in Arabidopsis, AtIAA27 was most similar and the likely ortholog to HaIAA27 (Fig. S1 and SI Materials and Methods).

We next investigated the amino acid sequences involved in the interaction between HaIAA27 and HaHSFA9. Different HaHSFA9 sequences fused to the GAL4 DNA-binding domain (GBD, bait constructs) were tested for yeast two-hybrid interaction with distinct HaIAA27 sequences fused to GAL4 activation domain (GAD, prey constructs). Fig. 1 shows that the complete HaIAA27 protein fusion interacts not only with the bait construct used to screen the cDNA library (with C-terminal deleted HaHSFA9 sequences), but also with a different bait containing the complete HaHSFA9 sequence with three amino acid substitutions. These substitutions were required to render the full-length bait fusion transcription inactive in yeast cells. From the HSF side, deletion of the N-terminal extension or the DNA binding domain (DBD) still allowed interaction with the full-length HaIAA27 prey fusion. However, deletion of the oligomerization domain (OD) and sequences that link the OD and the DBD in HaHSFA9 abolished the two-hybrid interaction. From the Aux/IAA side, whereas N-terminal truncated proteins (lacking the conserved domains I and II) interacted with HaHSFA9, C-terminal truncated proteins (lacking the conserved domains III and IV) did not. In addition prey HaIAA27 fusion proteins containing only domain IV or domains I+II+III did not show two-hybrid interaction. In conclusion, HaHSFA9 sequences in the OD (1) used in HSF–HSF interactions (21) are required for interaction with HaIAA27. Furthermore, domains III and IV of HaIAA27 (used Aux/IAA-Aux/IAA and Aux/IAA–ARF interactions) (15–18) are also required for two-hybrid interaction with HaHSFA9.

Fig. 1.

HaHSFA9 and HaIAA27 interact in a yeast two-hybrid assay. (Right) Spotted onto selective medium we show strains containing the indicated bait and prey plasmids. (Left) representation of the different versions of the HaHSFA9 and HaIAA27 proteins fused to the GAL4 DNA binding domain (GBD) or to the GAL4 activation domain (GAD) in the bait and prey plasmids, respectively. The maps at the bottom help to identify the protein domains included in each tested combination. Numbers beside maps correspond to plasmid constructs described in SI Materials and Methods. Domains I, II, III, and IV in the complete HaIAA27 protein (HaIAA27WT) are indicated. We also depict the N-terminal extension (NH), DNA binding domain (DBD), linker region (Lnk), and oligomerization domain (OD) on the map for the mutant activation domain form of HaHSFA9 (HaHSFA9mAD) used in these experiments. The positions of amino acid substitutions in HaHSFA9mAD are also indicated (with X).

To provide evidence for a direct interaction between HaHSFA9 and HaIAA27, we performed in vitro pull-down assays using fusion proteins expressed in Escherichia coli. The findings shown in Fig. S2 demonstrate that the complete WT form of HaIAA27 interacts with HaHSFA9, either as the complete HSF or, more efficiently, as a N-terminal deletion conserving the OD, which in yeast was necessary for two-hybrid interaction between HaIAA27 and HaHSFA9 (Fig. 1).

We next tried to confirm in planta the predicted interaction between HaIAA27 and HaHSFA9 (Fig. 1 and Fig. S2). Preliminary findings suggested that HaIAA27 was very unstable in leaves. We obtained stabilized forms of HaIAA27 by introducing amino acid substitutions in domain II. These substitutions involve conserved proline residues in the central GWPPV “degron” motif that is required for auxin-induced degradation of different Aux/IAA proteins. Stability and subcellular localization of the mutant forms was analyzed using HaIAA27-GFP fusions. The WT and mutant fusion proteins were studied in agro-infiltrated N. benthamiana leaves. Results in Fig. 2A show that either deletion (as in HaIAA27ΔN) or mutation (as in mIIa and in mIIab) of domain II in HaIAA27 drastically increased accumulation of the respective fusion proteins. The analyzed mutant forms were primarily nuclear localized, as was the WT HaIAA27-GFP form. A subtle localization change within the nucleus was evident for the mIIab form, which showed a diffuse pattern compared with the speckle distribution of the unstable WT form. The ΔN form showed bipartite nuclear/cytosolic localization (Fig. 2B). Results from Fig. 2 A and B strongly indicated that HaIAA27 is very unstable in leaves, where its nuclear degradation would be auxin inducible. Additional findings support this suggestion, as coexpression of WT HaIAA27-GFP and TIR1 in N. benthamiana leaves further reduced the accumulation of the WT HaIAA27-GFP protein (Fig. 2C). From the results in Fig. 2, we selected mIIab as the best-stabilized HaIAA27 form that could be used for BiFC interaction experiments.

Fig. 2.

HaIAA27 is destabilized by domain-II/TIR1-dependent mechanisms in N. benthamiana leaves. (A) Accumulation level of the different HaIAA27:GFP fusions: HaIAA27WT (WT), HaIAA27mIIa (mIIa), HaIAA27mIIab (mIIab), and HaIAA27ΔN (ΔN). Pooled protein samples were used for Western detection (labeled 1 and 2). The NPTII protein was used as an internal standard. The two consecutive proline residues in the GWPPV degron motif of domain II (P₉₆ and P₉₇; see also Fig. S1) were mutated to SP or SL, respectively, in the HaIAA27mIIa and HaIAA27mIIab forms. Constructs maps are at the top. (B) Confocal microscopy localization of the HaIAA27:GFP fusions. The inset in the WT panel shows a nucleus under higher magnification with a thin line pointing to nuclear speckles observed only with this form. (Scale bars, 10 μm; Inset, 2 μm.) (C) TIR1 further reduced the accumulation level of the HaIAA27WT form. We compare results obtained with the WT form plasmid alone (WT) and coinfiltrated with a TIR1:HA:DsRed plasmid (WT+TIR1). The pooled samples analyzed for each combination are labeled 1–4. The different conditions used to detect the WT fusion in A and C, as well as for expression and detection of each fusion in B, are given in SI Materials and Methods.

HaHSFA9 is one of the TFs involved in the activation of specific shsp gene expression in developing sunflower seeds (refs. 1, 3, 5 and references therein). The HaHSFA9 protein accumulates at a similar level from 8 d postanthesis (dpa, mid-embryogenesis) to 22 dpa (late embryogenesis, 1). In contrast, the sHSP-CI proteins encoded by HaHSFA9 target genes accumulate only in late embryogenesis (at ≈25 dpa) (22). It is possible that HaHSFA9 is repressed by HaIAA27 during mid-zygotic embryogenesis and that this repression is relieved at later stages. We investigated this possibility by examining in vivo interaction between a stabilized version (mIIab) of HaIAA27 and HaHSFA9 in immature sunflower embryos (15 dpa). BiFC experiments were performed after bombardment with YFP^C and YFP^N protein fusions in plasmids derived from improved BiFC vectors that allow detection of weak protein–protein interactions (SI Materials and Methods). The results in Fig. 3A show that HaIAA27mIIab and HaHSFA9 indeed showed interaction in the nucleus of cells from 15-dpa sunflower embryos. Interestingly, the WT HaIAA27 form also showed a similar interaction (Fig. 3B), which was not observed with control combinations of the two empty YFP^C and YFP^N vectors (Fig. 3C). Our findings show in planta interaction between an Aux/IAA protein (HaIAA27) and a TF different from ARFs (HaHSFA9). In addition, the results in Fig. 3 indicate that the WT HaIAA27 fusion protein could be stabilized, at least to some extent, in immature zygotic embryos.

Fig. 3.

HaHSFA9 and HaIAA27 interact in immature sunflower embryos. Confocal microscopy images of 15-dpa embryo cells cobombarded with plasmid constructs that express YFP fusions of HaIAA27 and HaHSFA9. (A) BiFC interaction between HaIAA27mIIab and HaHSFA9. (B) BiFC interaction between HaIAA27WT and HaHSFA9. (C) Control combining the empty YFP^C and YFP^N vectors. The C-terminal (YFP^C) half of YFP was fused to the complete HaHSFA9 protein. The N-terminal half (YFP^N) was fused to the mIIab form of HaIAA27, and also to the WT form of HaIAA27. (Scale bars, 10 μm.)

If the interaction between HaIAA27 and HaHSFA9 is functional in plant cells, HaIAA27 could repress HaHSFA9 in a similar way as for the repression of ARFs by Aux/IAA proteins. This was tested first by analyzing the effect of HaIAA27 on transient transcriptional activation of the Hahsp17.6G1 promoter (G1) by HaHSFA9 in sunflower embryos (Fig. 4). We used a minimal promoter, −126(G1), which includes HSE (heat-shock response elements, binding sites for HSFs), but does not include AuxREs (auxin-responsive elements, binding sites for ARFs). We observed a clear repression effect of the WT HaIAA27 form in immature embryos (15 dpa), but no sign of repression in older (20 dpa) embryos (Fig. 4A and 4B, respectively). These findings agree with detection of BiFC interaction between HaHSFA9 and HaIAA27 in 15-dpa embryos (Fig. 3). The results shown in Fig. 4 indicate that HaIAA27 represses HaHSFA9 in immature zygotic embryos, and that the repression is relieved by 20 dpa. A connection of auxin with such relief was investigated. The accumulation of LUC fusions of the WT and mIIab forms of HaIAA27 were analyzed in 15-dpa embryos treated with and without exogenous IAA (Fig. 5). We could detect both fusion forms, the fully stabilized mIIab protein still at higher level (≈20×) than the WT form. However, only the WT form reduced its accumulation level in response to the IAA treatment. These findings are in agreement with our analysis of HaIAA27 stability in leaves (Fig. 2). Auxin/TIR1-dependent destabilization of the WT form in maturing embryos could thus explain, at least in part, the repression relief. To further investigate possible connection with auxins, we analyzed the activity of the DR5:LUC reporter gene in bombarded sunflower embryos. Results in Fig. S3 show that LUC activity from the reporter gene increased about 2.61-fold between 15 and 20 dpa in sunflower embryos. These findings support that an increase in endogenous auxin content correlates with HaIAA27 repression relief.

Fig. 4.

HaIAA27 represses the transcriptional activation of the G1 promoter by HaHSFA9. (A) Transient assays performed with immature (15 dpa) sunflower embryos. The −126(G1) reporter gene was bombarded to 15-dpa embryos without effector plasmids (−) or with the indicated effector plasmid combination. Constructs maps are at the top. Mean Potinus luciferase (LUC) activity normalized with Renilla luciferase (RLUC) is plotted. Numbers in parentheses show the replicates analyzed per each combination. The occurrence of significant statistical differences is indicated with different bar shading. (B) Assays as in A performed with 20-dpa embryos. Effector plasmids used alone or in combination: 35S:HaHSFA9 (A9), 35S:HaIAA27WT (WT), 35S:HaIAA27ΔN (ΔN). The results of statistical analyses are detailed in Table S1.

Fig. 5.

Auxin treatments reduce the level of HaIAA27WT:LUC in 15-dpa sunflower embryos. We depict LUC fluorescence levels in embryos expressing an auxin reporter gene (DR5::luc), the HaIAA27mIIab:LUC (mIIab::luc), or HaIAA27WT:LUC (WT::luc) fusion with (+) and without (−) addition of 50 μM IAA in the medium. Statistical analyses and other symbols indicated as in Fig. 4.

That HaIAA27 may repress HSFA9 (and thus negatively affect target genes of HSFA9) was analyzed in transgenic tobacco. We overexpressed HaIAA27 from the seed-specific DS10 promoter and regulatory sequences (also used in ref. 3 to overexpress HaHSFA9) in the DS10:HaIAA27WT, DS10:HaIAA27mIIab, and DS10:HaIAA27ΔN plants. In seeds from plants with a single-integration event of the DS10:HaIAA27mIIab gene in homozygosis, HaIAA27 reduced the accumulation of seed sHSPs. The same was observed for DS10:HaIAA27ΔN seeds. In contrast, no such a reduction was observed in seeds from the DS10:HaIAA27WT lines (Fig. 6A). The observed reduction in the accumulation of seed sHSPs is similar to what was recently reported for loss of function of the HSFA9 program in transgenic tobacco using a dominant-negative form of HaHSFA9 (5). Thus, the observed reduction involved all of the CI sHSPs that accumulate in seeds and that are negatively affected by loss of function of HSFA9. This reduction was evident in the homozygous transgenic progeny (T₁) compared with nontransgenic sibling lines (Fig. 6A). The repression of HSFs (at least of HaHSFA9 and the endogenous HSFA9 in tobacco) by HaIAA27 could be thus observed not only in transient expression experiments (Fig. 4), but also inferred from the results obtained in transgenic seeds after stable transformation (Fig. 6A). The different forms of HaIAA27 expressed in tobacco seeds were epitope tagged with HA, which allowed us to determine that in mature seeds the WT form accumulated at a much lower level than the ΔN and mIIab forms (Fig. 6B).

Fig. 6.

Stabilized HaIAA27 forms reduce CI sHSP accumulation in transgenic tobacco seeds. (A) Maps of the DS10 chimeric genes used for seed-specific overexpression of the different forms. (Upper) HaIAA27WT (WT), HaIAA27mIIab (mIIab), and HaIAA27ΔN (ΔN) genes are depicted. Amino acid substitutions (labeled X) in the mIIab form are indicated as well as the position of Hemaglutinin (HA) tag. (Lower) Analysis of sHSP CI proteins in mature seeds. Two pairs (labeled 1 and 2) of sibling transgenic (T) and nontransgenic (NT) lines were analyzed. Seed-specific polypeptides are pointed by the arrow. (B) Relative levels of the WT, mIIab, and ΔN HaIAA27 forms in mature seeds (samples 1–3). The different forms were detected using antibodies against the HA tag. Twenty-five micrograms of total protein was used for the WT samples, and 2.5 μg for the mIIab and ΔN samples. Ponceau S (P) staining was used to verify protein loading.

The deduced repression could involve passive and/or active mechanisms. To explore these possibilities, we analyzed in DS10:HaIAA27ΔN seeds the effect of an internal deletion form of HaIAA27, HaIAA27ΔN, which lacks N-terminal domains I and II, but retains domains III and IV. However, HaIAA27ΔN contains HaIAA27 sequences sufficient for interaction with HaHSFA9 (Fig. 1). The overexpression of stabilized forms of Aux/IAAs in transgenic plants results in different auxin-related phenotypes, which generally are not observed with the WT forms (i.e., 23). In agreement with this, the DS10:HaIAA27WT seeds did not show abnormal germination and grew without visible defects after imbibition (Fig. 7A). In contrast, germination and early growth of the DS10:HaIAA27mIIab seeds was drastically impaired: seedlings having shorter and thicker hypocotyl and main roots. The DS10:HaIAA27ΔN seeds showed much milder defects and were similar as for DS10:HaIAA27WT (Fig. 7A). Thus, only the stabilized form containing the N-terminal region of HaIAA27 (including the domain I)—i.e., HaIAA27mIIab but not HaIAA27ΔN—impaired germination and seedling growth. These results are reminiscent of phenotypes caused by repression of ARFs by stabilized Aux/IAAs, which require domain I (i.e., ref. 23). Surprisingly, in the DS10:HaIAA27ΔN seeds, we detected a reduction of accumulation of CI sHSPs similar to that observed for the DS10:HaIAA27mIIab seeds (Fig. 6A). We could also show that the ΔN form of HaIAA27 was able to repress activation of the −126(G1) promoter by HaHSFA9 in mature (20 dpa) bombarded sunflower embryos (Fig. 4B). This would confirm the noncanonical repression mechanism inferred from the results in the DS10:HaIAA27ΔN transgenic plants. Thus, both HaIAA27mIIab and HaIAA27ΔN could repress the HSFA9 program with similar efficiency. This finding indicates that repression of HSFs by HaIAA27 might be different from the repression of ARFs by Aux/IAA proteins.

Fig. 7.

Transgenic lines that overexpress stabilized HaIAA27 forms show altered seed germination phenotypes. (A) The retarded germination of the DS10:HaIAA27mIIab (mIIab) seeds contrasts the normal and slightly altered germination of the DS10:HaIAA27WT (WT) or DS10:HaIAA27ΔN (ΔN) seeds, respectively. (B) Ethanol content after CDT. Seeds from WT, ΔN, mIIab, and A9M3 (5) lines are compared. Average results shown correspond to three (WT, ΔN, and mIIab) or two (A9M3) sibling pairs of transgenic (T) and nontransgenic (NT) plant lines analyzed in two to three independent experiments. Statistical analyses and other symbols indicated as in Fig. 4.

An expected consequence of down-regulation of HSFA9 in seeds is a reduction of their longevity, as determined by artificial aging procedures (5). Such an effect could not be directly demonstrated in the DS10:HaIAA27mIIab seeds, because these seeds already showed poor and delayed germination without aging compared with DS10:HaIAA27WT seeds (Fig. 7A). However, increased ethanol production after natural or artificial aging indicates reduction of seed longevity (24). Results in Fig. 7B show that ethanol production in artificially aged DS10:HaIAA27mIIab and DS10:HaIAA27ΔN seeds, but not in DS10:HaIAA27WT seeds, is similar to that in seeds from the strongest HSFA9 loss of function lines (the A9M3 lines) (5). Furthermore, the DS10:HaIAA27ΔN seeds deteriorate in a similar way as the A9M3 seeds, both showing poor germination only after artificial aging (Fig. S4). These findings further support that the repressive effect of HaIAA27 in the DS10:HaIAA27mIIab and DS10:HaIAA27ΔN seeds involves HSFA9.

Discussion

Our findings strongly indicate that HSFs expressed in zygotic embryos of seeds, such as HaHSFA9 (1), are unique targets of Aux/IAA protein repression. Our findings support a unique auxin-response pathway. Through this pathway, auxin may mediate transcriptional responses by a mechanism not involving ARF repression by Aux/IAAs. In the HSF auxin-response pathway, mechanisms would be similar, but not identical, as in the interaction and repression of ARFs by Aux/IAAs.

HaIAA27 and HaHSFA9 mRNAs were repeatedly cloned from the same cDNA library (1), and thus are coexpressed in immature zygotic embryos of sunflower. Furthermore, a WT HaIAA27 protein interacts with (Fig. 3) and represses HaHSFA9 in these embryos, but not later in mature embryos (Fig. 4). HaHSFA9 and the seed sHSP CI proteins, which are encoded by target genes of HaHSFA9, have different accumulation patterns during sunflower embryogenesis. In fact, both the sHSP CI mRNA and protein (22) accumulate much later than in the case of HaHSFA9, which is a highly abundant protein in immature embryos, accumulating at a similar high level from as early as 8 dpa (1). This could be explained if HaIAA27 repressed HaHSFA9 until at least 15 dpa, fitting our transient expression analyses of the repression in sunflower embryos (Fig. 4). The results obtained upon seed-specific overexpression of the HaIAA27WT, mIIab, and ΔN forms (Fig. 6A) suggest that HaIAA27 also represses transcription activation by the HSFA9 ortholog tobacco gene. Only overexpression of the mIIab and ΔN forms caused a substantial reduction of the accumulation of specific sHSP proteins (Fig. 6A). These proteins were identified as encoded by targets genes of HSFA9, by means of gain (3, 4) and loss of function (5). A functional connection between HaIAA27 and the HSFA9 genetic program is additionally supported by the observed increase of ethanol production after aging of the DS10:HaIAA27mIIab and DS10:HaIAA27ΔN seeds (Fig. 7B). Ethanol production by these aged seeds was higher than of DS10:HaIAA27WT seeds and similar to that of aged DS10:HaHSF9-SRDX (A9M3) seeds (5). Because ethanol production after seed aging indicates reduced longevity (24), we conclude that the inferred repression of the HSFA9 program by the mIIab and ΔN forms had similar effects as loss of function of HSFA9 in the A9M3 seeds (5). These findings suggest that repression by these forms could involve HSFA9 and other redundant HSFs, as proposed for the dominant-negative effect of HaHSF9-SRDX.

Domains III and IV of HaIAA27 (used for Aux/IAA-Aux/IAA and Aux/IAA–ARF interactions) (15–18) are also required for yeast two-hybrid interaction between HaHSFA9 and HaIAA27 (Fig. 1). As mentioned previously, we inferred that a HaIAA27 form without domains I and II (HaIAA27ΔN)—that is, able to interact with HaHSFA9—might repress HSFA9 in transgenic tobacco. Thus at least in embryos, the repression of HSFA9 by HaIAA27 could involve a passive mechanism (i.e., by interacting with the OD of the HSF, HaIAA27ΔN could have impaired the oligomerization and transcriptional activity of HSFA9). The repressive capacity of HaIAA27ΔN was confirmed in bombarded embryos (Fig. 4B). Active repression by HaIAA27ΔN would not be possible, at least by similar mechanisms as for the repression of ARFs involving domain I of Aux/IAAs and recruitment of TPL corepressors (7, 14). The results of Fig. 6A for HaIAA27ΔN contrast those of Fig. 7A for the same truncated form. There, the germination effects—interpreted as the consequence of the accumulation of stabilized HaIAA27 forms—required the presence of the N-terminal region of the protein (including domain I); these effects are observed only with the mIIab form. In vegetative organs the repressive mechanisms of HaIAA27 are therefore different; the observed phenotype fits with the involvement of classical repression of ARFs (7, 14, 23).

The accumulation of the HaIAA27 protein is likely regulated by auxin, which would induce its TIR1-dependent degradation in the proteasome. The HaIAA27 protein would show highest stability (accumulation level) in immature zygotic embryos, and it would be destabilized later on during seed maturation. This might lead to the alleviation of repression of HSFA9 in mature embryos. Our proposal is supported by analyses performed with different fusion proteins. For example, HaIAA27-GFP proteins were analyzed by transient expression in N. benthamiana leaves, where the WT form accumulated to a barely detectable level, whereas the auxin-resistant mIIab form accumulated to very high levels. This finding and the TIR1-induced reduction of the accumulation of WT HaIAA27 point to auxin/TIR1-induced destabilization of HaIAA27 (Fig. 2 A and C). Furthermore, the level of a WT form of HaIAA27 fused to LUC, but not that of a similar mIIab form, was reduced by auxin treatments in sunflower embryos (Fig. 5). Increased DR5:LUC reporter levels in maturing sunflower embryos would fit the proposed auxin link (Fig. S3). Other analyses—performed with HA-tagged proteins in tobacco—suggests that HaIAA27 is destabilized in maturing seeds (Fig. 6B). The suggested differential stabilization of HaIAA27 in immature seed embryos would be unique among Aux/IAAs. There are very few precedent studies where a given Aux/IAA protein showed different stability in distinct organ/tissues, or at different developmental stages. Interestingly, in rice flag leaves, the IAA27 protein gradually accumulated from 0 to 14 d after anthesis, reaching peak accumulation at day 21 and then decreasing to its lowest level by day 28 (25). This would indicate that IAA27 proteins could show differential stability in organs other than in seeds.

HSF/auxin-response pathways similar to what are proposed here for HaHSFA9 and HaIAA27 could help to explain some observations in the literature. For example, the reported auxin-induced accumulation of shsp mRNAs in embryos (26) may rely on HSFs and Aux/IAAs. Gravitropic defects and other altered responses to auxin that are observed with rha1, an AtHSFA4 null-mutant (27), could also be explained by HSF–Aux/IAA interactions. Similar interactions might explain connections of auxin responses with thermotolerance (28). HSF–Aux/IAA interactions could also clarify links of auxin with responses to high temperature (29). Finally, the data in Fig. 7B and Fig. S4, where the expression of stabilized forms of HaIAA27 leads to reduced seed longevity, would fit with previous observations that link reduction of seed auxin and loss of longevity (30). In addition, the Arabidopsis HSFA9 gene is a direct target of ABI3 (2), a transcription factor that is auxin inducible and links auxin and ABA responses in seeds (31, 32). Thus, auxin would have positive transcriptional effects on HSFA9. Repression alleviation of Arabidopsis HSFA9 by a mechanism similar to that proposed here could reinforce auxin responses in seeds.

The interaction between HaIAA27 and a HSF demonstrates a unique type of combinatorial transcriptional control in plant seeds. This interaction involves an Aux/IAA protein and TFs distinct from ARFs. Myb TFs as MYB77 are also involved in auxin responses, but it is not known if these TFs interact with Aux/IAAs (33). We infer that HSFs as HaHSFA9 and HaIAA27 mediate some auxin responses of developing seeds in connection with longevity. These HSFs would be a molecular link between auxins and sHSP seed expression. Our findings that HSFs are also targets of Aux/IAA repression widen possible interpretations of plant auxin responses. Phenotypes involving Aux/IAA loss or gain of function should not be interpreted hereafter solely based in potential Aux/IAA–ARF genetic interactions.

Materials and Methods

The construction of the different plasmids used in this study, the analyses in N. benthamiana, yeast two-hybrid, and additional details for the procedures outlined here are described in SI Materials and Methods. The immature (14 dpa) sunflower embryo cDNA library (1) was screened by two-hybrid in the PJ69-4A yeast strain, using as a bait plasmid pGBT9-HaHSFA9ΔAD. The transient assays using bombarded sunflower embryos, at 15 or 20 dpa, were performed essentially as previously described (1). For BiFC assays, the embryos were similarly bombarded. Seed longevity was determined from germination (5) or from production of ethanol (25) after controlled deterioration (CDT). Ethanol was determined from 10-mg seed samples placed in 0.5 mL water at 20 °C for 48 h, immediately after CDT for 24 h at 50 °C. Ethanol content was measured using the K-ETOH enzymatic determination kit (Megazyme). Statistical analyses were as described (5).

Acknowledgments

This work was supported by the Spanish Ministry of Science and Innovation-FEDER Grants BIO2005-0949 and BIO2008-00634 and the Andalusian Regional Government Grant BIO148. Support was also provided by the Consejo Superior de Investigaciones Científicas I3P program (to R.C.).

Footnotes

²To whom correspondence should be addressed. E-mail: fraga{at}cica.es.
Author contributions: J.J. designed research; R.C., J.M.E., P.P.-D., and C.A. performed research; P.P.-D., C.A., and J.J. analyzed data; and J.J. wrote the paper.
The authors declare no conflict of interest.
↵*This Direct Submission article had a prearranged editor.
Data deposition: The sequence reported in this paper has been deposited in the deposited in the GenBank database (accession no. FR669188).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1014856107/-/DCSupplemental.

Freely available online through the PNAS open access option.

References

↵
1. Almoguera C,
2. et al.
(2002) A seed-specific heat-shock transcription factor involved in developmental regulation during embryogenesis in sunflower. J Biol Chem 277:43866–43872.
OpenUrl Abstract/FREE Full Text
↵
1. Kotak S,
2. Vierling E,
3. Bäumlein H,
4. von Koskull–Döring P
(2007) A novel transcriptional cascade regulating expression of heat stress proteins during seed development of Arabidopsis. Plant Cell 19:182–195.
OpenUrl Abstract/FREE Full Text
↵
1. Prieto–Dapena P,
2. Castaño R,
3. Almoguera C,
4. Jordano J
(2006) Improved resistance to controlled deterioration in transgenic seeds. Plant Physiol 142:1102–1112.
OpenUrl Abstract/FREE Full Text
↵
1. Prieto–Dapena P,
2. Castaño R,
3. Almoguera C,
4. Jordano J
(2008) The ectopic overexpression of a seed-specific transcription factor, HaHSFA9, confers tolerance to severe dehydration in vegetative organs. Plant J 54:1004–1014.
OpenUrl CrossRef PubMed
↵
1. Tejedor–Cano J,
2. et al.
(2010) Loss of function of the HSFA9 seed longevity program. Plant Cell Environ 33:1408–1417.
OpenUrl PubMed
↵
1. Dreher KA,
2. Brown J,
3. Saw RE,
4. Callis J
(2006) The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness. Plant Cell 18:699–714.
OpenUrl Abstract/FREE Full Text
↵
1. Tiwari SB,
2. Wang XJ,
3. Hagen G,
4. Guilfoyle TJ
(2001) AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Plant Cell 13:2809–2822.
OpenUrl Abstract/FREE Full Text
↵
1. Leyser O
(2002) Molecular genetics of auxin signaling. Annu Rev Plant Biol 53:377–398.
OpenUrl CrossRef PubMed
↵
1. Kieffer M,
2. Neve J,
3. Kepinski S
(2010) Defining auxin response contexts in plant development. Curr Opin Plant Biol 13:12–20.
OpenUrl CrossRef PubMed
↵
1. Chapman EJ,
2. Estelle M
(2009) Mechanism of auxin-regulated gene expression in plants. Annu Rev Genet 43:265–285.
OpenUrl CrossRef PubMed
↵
1. Dharmasiri N,
2. Dharmasiri S,
3. Estelle M
(2005) The F-box protein TIR1 is an auxin receptor. Nature 435:441–445.
OpenUrl CrossRef PubMed
↵
1. Kepinski S,
2. Leyser O
(2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435:446–451.
OpenUrl CrossRef PubMed
↵
1. Tan X,
2. et al.
(2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446:640–645.
OpenUrl CrossRef PubMed
↵
1. Szemenyei H,
2. Hannon M,
3. Long JA
(2008) TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science 319:1384–1386.
OpenUrl Abstract/FREE Full Text
↵
1. Tatematsu K,
2. et al.
(2004) MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 16:379–393.
OpenUrl Abstract/FREE Full Text
↵
1. Weijers D,
2. et al.
(2006) Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis. Dev Cell 10:265–270.
OpenUrl CrossRef PubMed
↵
1. Hardtke CS,
2. et al.
(2004) Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development 131:1089–1100.
OpenUrl Abstract/FREE Full Text
↵
1. Muto H,
2. et al.
(2006) Fluorescence cross-correlation analyses of the molecular interaction between an Aux/IAA protein, MSG2/IAA19, and protein-protein interaction domains of auxin response factors of arabidopsis expressed in HeLa cells. Plant Cell Physiol 47:1095–1101.
OpenUrl Abstract/FREE Full Text
↵
1. Colón-Carmona A,
2. Chen DL,
3. Yeh KC,
4. Abel S
(2000) Aux/IAA proteins are phosphorylated by phytochrome in vitro. Plant Physiol 124:1728–1738.
OpenUrl Abstract/FREE Full Text
↵
1. Padmanabhan MS,
2. Shiferaw H,
3. Culver JN
(2006) The tobacco mosaic virus replicase protein disrupts the localization and function of interacting Aux/IAA proteins. Mol Plant Microbe Interact 19:864–873.
OpenUrl CrossRef PubMed
↵
1. Scharf KD,
2. et al.
(1998) The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules. Mol Cell Biol 18:2240–2251.
OpenUrl Abstract/FREE Full Text
↵
1. Coca MA,
2. Almoguera C,
3. Jordano J
(1994) Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: Localization and possible functional implications. Plant Mol Biol 25:479–492.
OpenUrl CrossRef PubMed
↵
1. Sato A,
2. Yamamoto KT
(2008) Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis. Physiol Plant 133:397–405.
OpenUrl CrossRef PubMed
↵
1. Woodstock LW,
2. Taylorson RB
(1981) Ethanol and acetaldehyde in imbibing soybean seeds in relation to deterioration. Plant Physiol 67:424–428.
OpenUrl Abstract/FREE Full Text
↵
1. Li ZW,
2. et al.
(2009) Differential expression and function analysis of proteins in flag leaves of rice during grain filling. Acta Agron Sin 35:132–139.
OpenUrl
↵
1. Kitamiya E,
2. Suzuki S,
3. Sano T,
4. Nagata T
(2000) Isolation of two genes that were induced upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentrations of 2,4-D. Plant Cell Rep 19:551–557.
OpenUrl CrossRef
↵
1. Fortunati A,
2. Piconese S,
3. Tassone P,
4. Ferrari S,
5. Migliaccio F
(2008) A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor. J Exp Bot 59:1363–1374.
OpenUrl Abstract/FREE Full Text
↵
1. Ludwig-Müller J,
2. Krishna P,
3. Forreiter C
(2000) A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress. Plant Physiol 123:949–958.
OpenUrl Abstract/FREE Full Text
↵
1. Gray WM,
2. Ostin A,
3. Sandberg G,
4. Romano CP,
5. Estelle M
(1998) High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis. Proc Natl Acad Sci USA 95:7197–7202.
OpenUrl Abstract/FREE Full Text
↵
1. Sreeramulu N
(1983) Auxins, inhibitors and phenolics in bambarranut seeds (Voandzeia subterranea Thouars) in relation to loss of viability during storage. Ann Bot (Lond) 51:209–216.
OpenUrl Abstract/FREE Full Text
↵
1. Brady SM,
2. Sarkar SF,
3. Bonetta D,
4. McCourt P
(2003) The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant J 34:67–75.
OpenUrl CrossRef PubMed
↵
1. Belin C,
2. Megies C,
3. Hauserová E,
4. Lopez-Molina L
(2009) Abscisic acid represses growth of the Arabidopsis embryonic axis after germination by enhancing auxin signaling. Plant Cell 21:2253–2268.
OpenUrl Abstract/FREE Full Text
↵
1. Shin R,
2. et al.
(2007) The Arabidopsis transcription factor MYB77 modulates auxin signal transduction. Plant Cell 19:2440–2453.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 107 (50)

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Article Classifications

Biological Sciences
Plant Biology

[1] ↵
Almoguera C,
et al.
(2002) A seed-specific heat-shock transcription factor involved in developmental regulation during embryogenesis in sunflower. J Biol Chem 277:43866–43872.
OpenUrl Abstract/FREE Full Text

[2] Almoguera C,

[3] et al.

[4] ↵
Kotak S,
Vierling E,
Bäumlein H,
von Koskull–Döring P
(2007) A novel transcriptional cascade regulating expression of heat stress proteins during seed development of Arabidopsis. Plant Cell 19:182–195.
OpenUrl Abstract/FREE Full Text

[5] Kotak S,

[6] Vierling E,

[7] Bäumlein H,

[8] von Koskull–Döring P

[9] ↵
Prieto–Dapena P,
Castaño R,
Almoguera C,
Jordano J
(2006) Improved resistance to controlled deterioration in transgenic seeds. Plant Physiol 142:1102–1112.
OpenUrl Abstract/FREE Full Text

[10] Prieto–Dapena P,

[11] Castaño R,

[12] Almoguera C,

[13] Jordano J

[14] ↵
Prieto–Dapena P,
Castaño R,
Almoguera C,
Jordano J
(2008) The ectopic overexpression of a seed-specific transcription factor, HaHSFA9, confers tolerance to severe dehydration in vegetative organs. Plant J 54:1004–1014.
OpenUrl CrossRef PubMed

[15] Prieto–Dapena P,

[16] Castaño R,

[17] Almoguera C,

[18] Jordano J

[19] ↵
Tejedor–Cano J,
et al.
(2010) Loss of function of the HSFA9 seed longevity program. Plant Cell Environ 33:1408–1417.
OpenUrl PubMed

[20] Tejedor–Cano J,

[21] et al.

[22] ↵
Dreher KA,
Brown J,
Saw RE,
Callis J
(2006) The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness. Plant Cell 18:699–714.
OpenUrl Abstract/FREE Full Text

[23] Dreher KA,

[24] Brown J,

[25] Saw RE,

[26] Callis J

[27] ↵
Tiwari SB,
Wang XJ,
Hagen G,
Guilfoyle TJ
(2001) AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Plant Cell 13:2809–2822.
OpenUrl Abstract/FREE Full Text

[28] Tiwari SB,

[29] Wang XJ,

[30] Hagen G,

[31] Guilfoyle TJ

[32] ↵
Leyser O
(2002) Molecular genetics of auxin signaling. Annu Rev Plant Biol 53:377–398.
OpenUrl CrossRef PubMed

[33] Leyser O

[34] ↵
Kieffer M,
Neve J,
Kepinski S
(2010) Defining auxin response contexts in plant development. Curr Opin Plant Biol 13:12–20.
OpenUrl CrossRef PubMed

[35] Kieffer M,

[36] Neve J,

[37] Kepinski S

[38] ↵
Chapman EJ,
Estelle M
(2009) Mechanism of auxin-regulated gene expression in plants. Annu Rev Genet 43:265–285.
OpenUrl CrossRef PubMed

[39] Chapman EJ,

[40] Estelle M

[41] ↵
Dharmasiri N,
Dharmasiri S,
Estelle M
(2005) The F-box protein TIR1 is an auxin receptor. Nature 435:441–445.
OpenUrl CrossRef PubMed

[42] Dharmasiri N,

[43] Dharmasiri S,

[44] Estelle M

[45] ↵
Kepinski S,
Leyser O
(2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435:446–451.
OpenUrl CrossRef PubMed

[46] Kepinski S,

[47] Leyser O

[48] ↵
Tan X,
et al.
(2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446:640–645.
OpenUrl CrossRef PubMed

[49] Tan X,

[50] et al.

[51] ↵
Szemenyei H,
Hannon M,
Long JA
(2008) TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science 319:1384–1386.
OpenUrl Abstract/FREE Full Text

[52] Szemenyei H,

[53] Hannon M,

[54] Long JA

[55] ↵
Tatematsu K,
et al.
(2004) MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 16:379–393.
OpenUrl Abstract/FREE Full Text

[56] Tatematsu K,

[57] et al.

[58] ↵
Weijers D,
et al.
(2006) Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis. Dev Cell 10:265–270.
OpenUrl CrossRef PubMed

[59] Weijers D,

[60] et al.

[61] ↵
Hardtke CS,
et al.
(2004) Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development 131:1089–1100.
OpenUrl Abstract/FREE Full Text

[62] Hardtke CS,

[63] et al.

[64] ↵
Muto H,
et al.
(2006) Fluorescence cross-correlation analyses of the molecular interaction between an Aux/IAA protein, MSG2/IAA19, and protein-protein interaction domains of auxin response factors of arabidopsis expressed in HeLa cells. Plant Cell Physiol 47:1095–1101.
OpenUrl Abstract/FREE Full Text

[65] Muto H,

[66] et al.

[67] ↵
Colón-Carmona A,
Chen DL,
Yeh KC,
Abel S
(2000) Aux/IAA proteins are phosphorylated by phytochrome in vitro. Plant Physiol 124:1728–1738.
OpenUrl Abstract/FREE Full Text

[68] Colón-Carmona A,

[69] Chen DL,

[70] Yeh KC,

[71] Abel S

[72] ↵
Padmanabhan MS,
Shiferaw H,
Culver JN
(2006) The tobacco mosaic virus replicase protein disrupts the localization and function of interacting Aux/IAA proteins. Mol Plant Microbe Interact 19:864–873.
OpenUrl CrossRef PubMed

[73] Padmanabhan MS,

[74] Shiferaw H,

[75] Culver JN

[76] ↵
Scharf KD,
et al.
(1998) The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules. Mol Cell Biol 18:2240–2251.
OpenUrl Abstract/FREE Full Text

[77] Scharf KD,

[78] et al.

[79] ↵
Coca MA,
Almoguera C,
Jordano J
(1994) Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: Localization and possible functional implications. Plant Mol Biol 25:479–492.
OpenUrl CrossRef PubMed

[80] Coca MA,

[81] Almoguera C,

[82] Jordano J

[83] ↵
Sato A,
Yamamoto KT
(2008) Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis. Physiol Plant 133:397–405.
OpenUrl CrossRef PubMed

[84] Sato A,

[85] Yamamoto KT

[86] ↵
Woodstock LW,
Taylorson RB
(1981) Ethanol and acetaldehyde in imbibing soybean seeds in relation to deterioration. Plant Physiol 67:424–428.
OpenUrl Abstract/FREE Full Text

[87] Woodstock LW,

[88] Taylorson RB

[89] ↵
Li ZW,
et al.
(2009) Differential expression and function analysis of proteins in flag leaves of rice during grain filling. Acta Agron Sin 35:132–139.
OpenUrl

[90] Li ZW,

[91] et al.

[92] ↵
Kitamiya E,
Suzuki S,
Sano T,
Nagata T
(2000) Isolation of two genes that were induced upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentrations of 2,4-D. Plant Cell Rep 19:551–557.
OpenUrl CrossRef

[93] Kitamiya E,

[94] Suzuki S,

[95] Sano T,

[96] Nagata T

[97] ↵
Fortunati A,
Piconese S,
Tassone P,
Ferrari S,
Migliaccio F
(2008) A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor. J Exp Bot 59:1363–1374.
OpenUrl Abstract/FREE Full Text

[98] Fortunati A,

[99] Piconese S,

[100] Tassone P,

[101] Ferrari S,

[102] Migliaccio F

[103] ↵
Ludwig-Müller J,
Krishna P,
Forreiter C
(2000) A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress. Plant Physiol 123:949–958.
OpenUrl Abstract/FREE Full Text

[104] Ludwig-Müller J,

[105] Krishna P,

[106] Forreiter C

[107] ↵
Gray WM,
Ostin A,
Sandberg G,
Romano CP,
Estelle M
(1998) High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis. Proc Natl Acad Sci USA 95:7197–7202.
OpenUrl Abstract/FREE Full Text

[108] Gray WM,

[109] Ostin A,

[110] Sandberg G,

[111] Romano CP,

[112] Estelle M

[113] ↵
Sreeramulu N
(1983) Auxins, inhibitors and phenolics in bambarranut seeds (Voandzeia subterranea Thouars) in relation to loss of viability during storage. Ann Bot (Lond) 51:209–216.
OpenUrl Abstract/FREE Full Text

[114] Sreeramulu N

[115] ↵
Brady SM,
Sarkar SF,
Bonetta D,
McCourt P
(2003) The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant J 34:67–75.
OpenUrl CrossRef PubMed

[116] Brady SM,

[117] Sarkar SF,

[118] Bonetta D,

[119] McCourt P

[120] ↵
Belin C,
Megies C,
Hauserová E,
Lopez-Molina L
(2009) Abscisic acid represses growth of the Arabidopsis embryonic axis after germination by enhancing auxin signaling. Plant Cell 21:2253–2268.
OpenUrl Abstract/FREE Full Text

[121] Belin C,

[122] Megies C,

[123] Hauserová E,

[124] Lopez-Molina L

[125] ↵
Shin R,
et al.
(2007) The Arabidopsis transcription factor MYB77 modulates auxin signal transduction. Plant Cell 19:2440–2453.
OpenUrl Abstract/FREE Full Text

[126] Shin R,

[127] et al.

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Repression by an auxin/indole acetic acid protein connects auxin signaling with heat shock factor-mediated seed longevity

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Repression by an auxin/indole acetic acid protein connects auxin signaling with heat shock factor-mediated seed longevity

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