Abstract
The gaseous hormone ethylene participates in many physiological processes of plants. It is well known that ethylene-inhibited root elongation involves basipetal auxin delivery requiring PIN2. However, the molecular mechanism how ethylene regulates PIN2 is not well understood. Here, we report that the ethylene-responsive HD-Zip gene HB52 is involved in ethylene-mediated inhibition of primary root elongation. Using biochemical and genetic analyses, we demonstrated that HB52 is ethylene-responsive and acts immediately downstream of EIN3. HB52 knock-down mutants are insensitive to ethylene in primary root elongation while the overexpression lines have dramatically shortened roots like ethylene treated plants. Moreover, HB52 upregulates PIN2, WAG1, and WAG2 by directly binding to their promoter, leading to an enhanced basipetal auxin delivery to the elongation zone and thus inhibiting root growth. Our work uncovers HB52 as an important crosstalk node between ethylene signaling and auxin transport in root elongation.
Introduction
Ethylene is a gaseous phytohormone which regulates a multitude of processes at trace levels. It is well known for triggering the shedding of leaves, the ripening of fruits, and the defense of plants. It also plays an indispensable role in root development (Alonso and Ecker, 1999; Grbić and Bleecker, 2003; Chaves and Mello-Farias, 2006; Ruzicka et al., 2007). Exogenous treatment with ethylene (C2H4) or its biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) leads to the inhibition of primary root elongation, the increase of primary root width, and the induction of ectopic root hairs (Masucci and Schiefelbein, 1996; Smalle and Van Der Straeten, 1997; Le et al., 2001). These three ethylene induced responses will promote soil penetration and greater anchorage on the ground.
Great advances in ethylene signaling pathway have been made in the past decade using genetic approaches in Arabidopsis (Merchante et al., 2013). In the absence of ethylene, the receptors and other related proteins recruit the Raf-like kinase CTR1 which phosphorylates the C-terminal end of EIN2, thus preventing it from translocating into the nucleus to stabilize the downstream transcription factors EIN3/EIL1. In the presence of ethylene, the hormone binds to the receptors thus inactivating CTR1, so the unphosphorylated C-terminal end of EIN2 can be cleaved and moves into the nucleus to stabilize EIN3/ EIL1 which will activate the downstream transcriptional cascade (Gao et al., 2003; Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012).
Intriguingly, mutants of auxin synthesis, signaling pathway or transport show aberrant responses to ethylene, indicating crosstalk between these two hormones. For example, mutations in auxin biosynthesis genes such as ASA1, ASB1, TAR1 and TAA1 exhibit ethylene-insensitive root phenotypes (Stepanova et al., 2005; Stepanova et al., 2008). YUC genes also play key roles in ethylene-mediated root response (Won et al., 2011). Mutants of AXR2/IAA7 and AXR3/IAA17 which encode transcription regulators in the auxin signal pathway exhibit insensitive root growth to ethylene (Alonso et al., 2003). PIN2 and AUX1, two of the auxin transport components are also involved in ethylene-mediated root response (Ruzicka et al., 2007).
Plants have a considerable number of transcription factors which play vital roles in the different development process. Among all the families of transcription factors, the HD-Zip family is unique to plant. These proteins display a singular combination of a homeodomain with a leucine zipper working as a dimerization motif. This family consists of 47 members and can be classified into four subfamilies (Ariel et al., 2007). ATHB1 participates in the determination of leaf cell fate, whereas ATHB13 and ATHB23 are involved in cotyledon and leaf development (Aoyama et al., 1995; Nakamura et al., 2006). HAT2 overexpression lines have a representative phenotype of auxin-overproducing mutants indicating a role in auxin-mediated development (Delarue et al., 1998; Sawa et al., 2002). PHV, PHB, and REV have similar functions during embryogenesis and leaf polarity determination (Prigge and Clark, 2006). ATHB10, ATML1, and PDF2 play important roles in cell fates establishment by regulating cell layer-specific gene expression (Abe et al., 2003). Although some proteins in this family have been studied well in the past few years, others still need further investigation.
In this study, we report an HD-Zip gene HB52 which is involved in ethylene-mediated primary root elongation. HB52 knock-down mutants are insensitive to ethylene in primary root elongation while HB52 overexpression lines have shortened roots similar to ethylene treated plants. Biochemical and genetic assays showed that HB52 is a direct target of EIN3. DR5:GUS in HB52 mutants showed altered auxin basipetal transport. Further analyses demonstrated that HB52 could directly regulate PIN2, WAG1, and WAG2. Moreover, a clear PIN1 and PIN3 apical polarity in the stele and PIN2 apical polarity in the cortex were observed in the HB52 overexpression line. Our results indicate that HB52 plays a vital role in the inhibition of ethylene-induced primary root growth in Arabidopsis and acts as the crosstalk node between ethylene and auxin signaling pathways in primary root elongation.
Results
Expression pattern and subcellular localization of HB52
To investigate the expression pattern of HB52, we detected the transcription level of HB52 in different tissues of 4-week old plants by quantitative RT–PCR. The strongest expression was observed in roots followed by stem and rosette leaves (Figure 1A). To further confirm this result, we generated HB52pro:GUS transgenic lines. Histochemical analysis of the transgenic lines showed that HB52 promoter-driven GUS reporter was primarily expressed in the root tip and hypocotyl base of 4-day old young seedlings (Figure 1B, C and D). In 10-day old seedlings, GUS staining was mainly observed in roots and petiole of rosette leaves (Figure 1E). In mature plants, GUS staining was only found in roots (Figure 1F).
To investigate the subcellular localization of HB52, we generated 35S:HB52-GFP transgenic lines. Clear fluorescence was observed in the nucleus under confocal laser scanning microscope (Figure 1G). The nucleus localization of HB52 is in coincidence with its function as a transcription factor.
HB52 is responsive to ethylene, which depends on ethylene signaling
To confirm whether HB52 is regulated by ethylene and determine its position in ethylene signaling pathway, we detected the transcript level of HB52 in the wild type (Col-0) and ethylene signaling mutants using quantitative RT–PCR. HB52 was upregulated by exogenous ACC (Figure 2A). Moreover, HB52 was down regulated in ethylene signaling-blocked mutants ein2-5 and ein3-1eil1 and upregulated in ethylene signaling-enhanced mutants 35S:EIN3-GFP and ctr1-1 without or with exogenous ACC (Figure 2A). To confirm this, we introduced HB52pro:GUS into ein2-5, ein3-1eil1, 35S:EIN3-GFP and ctr1-1 background, respectively. The GUS staining of HB52pro:GUS was lighter in ein2-5 and ein3-1eil1 background while darker in 35S:EIN3-GFP and ctr1-1 background when compared with HB52pro:GUS without or with exogenous ACC (Figure 2B and 2C). These results indicate that HB52 acts downstream of EIN3 and EIL1.
HB52 regulates primary root elongation in response to ethylene
To study the role of HB52 in root elongation in response to ethylene, we obtained the mutant CS909234 with a T-DNA insertion in the promoter of HB52 from ABRC (Figure S1) and generated an estradiol-induced RNAi line RNAi-6. For clarity, the mutant CS909234 is renamed hb52. The transcript level of HB52 in hb52 and RNAi-6 was significantly reduced compared with that of the wild type (Figure 3A). Meanwhile, we tried to generate HB52 overexpression lines driven by 35S promoter but the transgenic plants failed to set seeds due to aberrant development of flowers (data not shown). Therefore, we generated HB52 overexpression lines driven by estradiol-inducible promoter instead. The transcript level of HB52 in three representative overexpression lines OX11-5, OX35-2, OX14-1 increased 30, 250 and 870 fold, respectively (Figure 3A). The relative primary root elongation of the three overexpression lines decreased to 87%, 66%, 43% of the wild type after induction (Figure S2B, left panel). Clearly, the primary root length is negatively correlated with HB52 expression level.
If germinated on MS medium with estradiol directly, the overexpression lines OX14-1 exhibited yellow colored cotyledon that might be caused by the high expression level of HB52 (Figure S2A). To test the response of HB52 mutants to ethylene and avoid the influence of yellow colored cotyledon, we germinated the seeds on MS medium for 3 days and then transferred the seedlings to MS medium with estradiol for another 3 days to induce gene expression. Afterwards, the seedlings were transferred to MS medium with estradiol supplemented with different concentration of ACC for 4 days to measure primary root elongation. Under 0 μM ACC, the primary root elongation of the knock-down mutants (RNAi-6 and hb52) was comparable to that of the wild type control while it was significantly reduced in the overexpression lines, among which the primary root elongation was negatively correlated with HB52 expression levels (Figure 3B, top panel). In response to ACC, the two HB52 knock-down lines and three overexpression lines were all less sensitive in root elongation compared with Col-0 (Figure 3B and C). Among the three overexpression lines, OX14-1 is the least sensitive line to ACC in root elongation followed by OX35-2 (Figure 3C). These results indicate that HB52 plays an important role in ethylene-inhibited primary root elongation.
In addition to altered primary root elongation, we observed other root phenotypes associated with varied HB52 expression levels, which include collapsed root meristem of the overexpression lines (Figure S2A) and altered root gravitropic response of the knock-down mutants and overexpression lines (Figure S3).
HB52 is a direct target of EIN3
We have previously shown that HB52 acts downstream of EIN3 and EIL1. So we next explored whether HB52 is a direct target of EIN3 and EIL1. Three putative EIN3-binding sites (EBS, TACAT or TTCAAA) were found in the promoter of HB52 (Konishi and Yanagisawa, 2008; Zhong et al., 2009; An et al., 2012; Li et al., 2013) (Figure 4A). We performed chromatin immunoprecipitation (ChIP) assays using 35S:EIN3-GFP and 35S:EIL1-GFP transgenic plants. Marked enrichment of the region containing cis2 site (TACAT) was detected in 35S:EIN3-GFP transgenic plants by ChIP–PCR assays (Figure 4B and 4C), indicating that EIN3 binds to this region in vivo. Furthermore, we conducted yeast-one-hybrid to determine whether EIN3 and EIL1 could directly bind to the EBS in the promoter of HB52. The result showed that EIN3 was able to bind to the cis2 site in the promoter of HB52 (Figure 4D). Taken together, these data suggest that HB52 is a direct target of EIN3.
To further confirm that HB52 acts downstream of EIN3, we crossed hb52 with 35S:EIN3-GFP and ctr1-1 separately. ctr1-1hb52 had the same point mutation with ctr1-1 and 35S:EIN3-GFPhb52 had the same expression level of EIN3 with 35S:EIN3-GFP (Figure S4A and S4B). HB52 expression level decreased in ctr1-1hb52 and 35S:EIN3-GFPhb52 (Figure S4C). The genetic assays showed that the roots of 35S:EIN3-GFPhb52 and ctr1-1hb52 are longer than that of 35S:EIN3-GFP and ctr1-1 without and with exogenous ACC (Figure 5A and 5B). This genetic evidence strongly supports that HB52 acts downstream of EIN3.
HB52 directly regulates PIN2, WAG1, and WAG2
We have noticed that HB52 knock-down lines and overexpression lines were all insensitive to ACC in root elongation. Obviously, HB52 plays an important role in ethylene-mediated root elongation. However, the underlying molecular mechanism is unknown. We introduced DR5-GUS reporter into hb52 and OX35-2 background by crossing to see if there is any change of auxin level in root tip. Both lines were confirmed by detecting the transcript level of HB52 (Figure 6A). Exogenous ACC clearly induces the expression of the DR5:GUS reporter in the elongation zone of the wild type but not in the hb52 background, indicating a blockage in auxin basipetal transport, while the expression of DR5:GUS is significantly reduced in the OX35-2 background without or with exogenous ACC (Figure 6B). Taken together, these results suggest that the basipetal transport of auxin is altered by HB52.
To investigate the role of HB52 in auxin basipetal transport, we examined the transcript level of PID, WAG1, WAG2 and other auxin transport related genes. As shown in Figure 6C, PIN2, WAG1, and WAG2 were downregulated in hb52 and upregulated in OX14-1. Moreover, several HB52 binding sites were found in promoters of PIN2, WAG1, and WAG2, suggesting that these three genes are direct targets of HB52.
To confirm that PIN2, WAG1, and WAG2 are direct targets of HB52, we demonstrated that HB52 was able to directly bind to at least one homeodomain binding site in the promoter of these three genes by using ChIP-PCR, yeast-one-hybrid, and EMSA (Figure 7, 8 and 9).
In order to confirm genetically that PIN2, WAG1, and WAG2 act downstream of HB52, we crossed the knockout mutants of PIN2 (pin2, CS8058), WAG1 (wag1, Salk_002056) and WAG2 (wag2, Salk_070240) with the HB52 overexpression line (OX35-2), respectively and confirmed the expression of HB52 (Figure 10A). The results in Figure 10B and 10C show that the primary roots of these hybrid lines are longer than the HB52 overexpression line with different degrees as predicted. These results suggest that HB52 depends on WAG1, WAG2, and PIN2 for its function in ethylene-mediated root elongation.
Discussion
Synergistic effects of auxin and ethylene have been extensively studied in the regulation of root elongation. Ethylene has been shown to increase auxin synthesis, auxin transport to the elongation zone, and auxin signaling at the root tip (Pickett et al., 1990; Alonso et al., 2003; Stepanova et al., 2005; Ruzicka et al., 2007; Swarup et al., 2007; Stepanova et al., 2008; Mao et al., 2016). The HD-Zip transcription factors are a unique family in plants and divided into 4 subfamilies I-IV mainly based on their structure and function. HB52 belongs to HD-ZIP I and has not been revealed for its role in plants. Members of this subfamily have been shown to be involved in abiotic stress response, ABA-mediated regulation, de-etiolation, and blue-light signaling (Ariel et al., 2007). In this study, we identified that ethylene-responsive HB52 acts directly downstream of EIN3 to affect auxin basipetal transport by regulating WAG1, WAG2, and PIN2.
It is known that HB52 can be upregulated by ethylene in the root in public data such as e-FP browser. A previous study also shows that EIN3, a master regulator of the ethylene signaling pathway, binds directly to the promoter of HB52 based on the data of EIN3 ChIP-Seq experiments (Chang et al., 2013). So we speculate that it may play a role in ethylene-mediated root regulation. To investigate its function, we first obtained the HB52 knock-down mutant and overexpression lines. Both HB52 knock-down mutant and overexpression lines are less sensitive to exogenous ACC in root elongation than wild type (Figure 3). Moreover, the primary roots of 35S:EIN3-GFPhb52 and ctr1-1 hb52 are longer than 35S:EIN3-GFP and ctr1-1 respectively, which further supports the role of HB52 in ethylene-mediated root elongation (Figure 5). Both ChIP-PCR and yeast-one-hybrid experiments confirm that EIN3 can bind to the promoter of HB52 (Figure 4), consistent with EIN3 ChIP-Seq data (Chang et al., 2013). The expression pattern of HB52pro:GUS reporter in transgenic lines also matches the function of HB52 in the root (Figure 1 and 2).
To investigate the specific mechanism by which HB52 controls root elongation. We introduced DR5:GUS reporter into hb52 and OX35-2 background. When treated with ACC, the staining of DR5:GUS in hb52 background showed a blockage in auxin basipetal transport (Figure 6B), which explains the insensitivity of knock-down lines to ethylene (Figure 3B and 3C). The staining of DR5:GUS is significantly reduced in OX35-2 background mainly due to the aberrant development of meristematic zone in the root (Figure 6B and S2A). This is the reason why overexpression lines are insensitive to ACC because ethylene-mediated root inhibition needs more auxin basipetal transport from the meristematic zone to the elongation zone (Ruzicka et al., 2007). The aberrant development of meristem is probably the cause of agravitropism (Figure S3) since auxin redistribution in the meristematic zone is of vital importance in regulating gravitropic response (Petrasek and Friml, 2009).
The root phenotype of HB52 overexpression lines is very similar to that of PID, WAG1 and WAG2 overexpression lines. Estradiol-induced overexpression of PID, WAG1 or WAG2 led to reduced DR5:GUS expression, loss of gravitropism and collapse of root meristem. It was reported that the collapsed root meristem can be rescued by NPA (Benjamins et al., 2001; Dhonukshe et al., 2010). We previously obtained 35S: HB52 lines with severe fertility problems (data not shown) just like the 35S: PID lines due to abnormal flower development (Benjamins et al., 2001). A frequent collapse of root meristem was observed in overexpression lines and can be rescued by NPA (Figure S2B, right panel). Considering the fact that auxin transport was altered in HB52 mutants and the overexpression lines had so many similarities with AGC3 kinase overexpression lines (Figure 6B, S2 and S3), we detected the transcript level of the genes related to auxin transport and found PIN2, WAG1, and WAG2 were downregulated in HB52 knock-down mutants and upregulated in overexpression lines (Figure 6C).
It has been shown that pin2/eir1 is insensitive to ethylene in root elongation and exogenous ACC upregulates the PIN2 expression of proPIN2:GUS and proPIN2:PIN2-GFP, indicating PIN2 is involved in the ethylene-mediated root inhibition. But PIN2 is not a direct target of EIN3 (Benjamins et al., 2001; Chang et al., 2013). The link between ethylene and PIN2 is still to be revealed. PID, WAG1, and WAG2 belong to the plant-specific AGCVIII family of kinases and work redundantly to instruct PIN apical polarity in root development. The most distal cells of the pidwag1wag2 root epidermis displayed basal localization of PIN2 as compared with its apical localization in wild type, while overexpression of these three genes leads to apically localized PIN1 in the root stele, PIN2 in the cortex and PIN4 in the root meristem (Dhonukshe et al., 2010). It has been demonstrated that PIN2 in the epidermis is responsible for auxin basipetal transport and required for root gravitropic response (Ruzicka et al., 2007). The root of HB52 knock-down mutant is agravitropic and show partly blocked auxin basipetal transport (Figure S3 and 6B) mainly due to the less apical localization of PIN2 in the epidermis caused by downregulation of PIN2, WAG1, and WAG2 (Figure 6C). By using yeast-one-hybrid, ChIP-PCR, EMSA, and genetic analyses, we further proved that PIN2, WAG1, and WAG2 are direct targets of HB52 in ethylene-mediated root inhibition (Figure 7, 8, 9 and 10).
Taken together, our results support a model where ethylene stabilizes EIN3 and upregulates HB52. HB52 then increases the expression of PIN2, WAG1, and WAG2. As a result, more auxin is transported to the elongation zone, leading to inhibition of root elongation.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as wild-type. A homozygous HB52 knock-down mutant CS909234 was ordered from Arabidopsis Biological Resource Center. The OX11-5, 35-2, 14-1, 18-4, RNAi-6, HB52pro:GUS, 35S:HB52-GFP, 35S:EIN3-GFP transgenic plants were obtained by Agrobacterium (C58C1) -mediated transformation using the Arabidopsis floral-dip method. For OX11-5, 35-2, 14-1 and 18-4, the HB52 coding sequence was amplified by pER8-HB52-P1 and pER8-HB52-P2 and cloned into pER8. For RNAi-6, about 200bp of the HB52 coding sequence was amplified by RNAi-P1 and RNAi-P2 and then by RNAi-P3 and RNAi-P4, both segments were cloned into phj33, and then shuttled it into the pER8. For HB52pro:GUS, the promoter of HB52 were amplified by GUS-HB52-P1 and GUS-HB52-P2 and cloned into pDONR207, and then shuttled it into the pCB308R. For 35S:HB52-GFP, the HB52 coding sequence without a stop codon were amplified by GFP-HB52-P1 and GFP-HB52-P2 and cloned into pDONR207, and then shuttled it into the pGWB5.
Several plant materials were previously described: ein2-5 (Alonso et al., 1999), ein3-1 eil1-1 (Alonso et al., 2003), ctr1-1 (Kieber et al., 1993), 35S:EIN3-GFP. HB52pro:GUSein2-5, HB52pro:GUSein3-1eil1, HB52pro:GUS35S:EIN3-GFP and HB52pro:GUSctr1-1 were crossed by HB52pro:GUS and ein2-5, ein3-1eil1, 35S:EIN3-GFP and ctr1-1 separately. ctr1-1 CS909234 and 35S:EIN3-GFP CS909234 were crossed by CS909234 with ctr1-1 and 35S:EIN3-GFP separately.
Arabidopsis seeds were surface sterilized in 10% bleach for 15 minutes and washed with distilled water for 6 times. Then the seeds were vernalized at 4°C for 3 days and vertically germinated on 1/2MS medium (Murashige and Skoog). If transferred to soil, all plants were grown under long day conditions (16-h light / 8-h dark) at 22–24°C.
Histochemical GUS staining and fluorescence observation
Histochemical GUS staining of transgenic plants was performed as previously described (Mao et al., 2016). Images were captured using an OLYMPUS IX81 microscope and HiROX (Japan) MX5040RZ.
Fluorescence observation of GFP transgenic plants was imaged using ZEISS710 confocal laser scanning microscope: 543nm for excitation and 620 nm for emission. Fluorescence observation of Propidium iodide (PI) stained transgenic plants. Seedlings were incubated in 10 mg/mL propidium iodide for 3 minutes and washed twice in water. The stained seedlings were imaged using ZEISS710 confocal laser scanning microscope: 488nm for excitation and 510 nm for emission.
RT-PCR and quantitative RT-PCR analysis
Total RNA was isolated using TRIzol reagent (Invitrogen) and reversed by TransScript RT kit (Invitrogen). Then cDNA was used for RT-PCR and quantitative RT-PCR. For RT–PCR analysis, the PCR products were amplified and examined on 2% agarose gel. Quantitative RT-PCR was performed on StepOne real-time PCR system using SYBR Premix Ex Taq II kit. Genes expression level was normalized by Ubiquitin5 (UBQ5, At3g62250).
Yeast-one-hybrid assay
Yeast one-hybrid assay was carried out as described previously (Mao et al., 2016). The coding sequence of proteins was cloned into pAD-GAL4-2.1 (AD vector) and the putative protein binding sites were cloned into pHIS2 (BD vector).
Starch granules staining
Starch granule staining was performed as described previously (Sabatini et al., 1999).
ChIP assay
ChIP assay was carried out as described previously (Cai et al., 2014).
EMSA assay
Competitors were commercially synthesized and free probes were synthesized with biotin labelled at the 5’ end. The coding sequence of HB52 was cloned into pMAL-C2 and the HB52-MBP fusion protein was expressed in Rosseta2 strain. EMSA assay was performed using LightShift™ EMSA Optimization and Control Kit (20148×) according to the manufacturer’s instructions.
Author Contributions
C.X. and Z.M. designed the experiments. Z.M., P.X., J.M., L.Y., Y.Y., and H.T. performed the experiments and data analyses. Z.M. wrote the manuscript. C.X supervised the project and revised the manuscript.
Acknowledgements
This study was supported by grants from NNSFC (grant no.91417306, 30830075), MOST (2012CB114304). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank ABRC for providing the mutant seeds.