JIPB

Journal of Integrative Plant Biology

Activated expression of AtEDT1/HDG11 promotes lateral root formation in Arabidopsis mutant edt1 by upregulating jasmonate biosynthesis Xiao-Teng Cai, Ping Xu, Yao Wang and Cheng-Bin Xiang*

Abstract Root architecture is crucial for plants to absorb water and nutrients. We previously reported edt1 (edt1D) mutant with altered root architecture that contributes significantly to drought resistance. However, the underlying molecular mechanisms are not well understood. Here we report one of the mechanisms underlying EDT1/HDG11conferred altered root architecture. Root transcriptome comparison between the wild type and edt1D revealed that the upregulated genes involved in jasmonate biosynthesis and signaling pathway were enriched in edt1D root, which were confirmed by quantitative RT-PCR. Further analysis showed that EDT1/HDG11, as a transcription factor, bound directly to the HD binding sites in the promoters of AOS, AOC3, OPR3, and OPCL1, which encode four key enzymes in JA biosynthesis. We found that the jasmonic acid level was significantly elevated in edt1D root compared with that in the wild type subsequently. In addition, more auxin accumulation was observed in the

lateral root primordium of edt1D compared with that of wild type. Genetic analysis of edt1D opcl1 double mutant also showed that HDG11 was partially dependent on JA in regulating LR formation. Taken together, overexpression of EDT1/HDG11 increases JA level in the root of edt1D by directly upregulating the expressions of several genes encoding JA biosynthesis enzymes to activate auxin signaling and promote lateral root formation.

INTRODUTION

Fattorini et al. 2009; Morquecho-Contreras et al. 2010; RayaGonzalez et al. 2012). The JA receptor CORONATINE INSENSITIVE 1 (COI1) plays an important role in JA-induced LR formation and LR positioning. JA regulation of postembryonic root development can be divided into auxin-dependent and independent mechanisms (Raya-Gonzalez et al. 2012). JA promotes LR formation by inducing ANTHRANILATE SYNTHASE alpha 1 (ASA1), which encodes auxin biosynthesis enzyme and can affect auxin transport (Sun et al. 2009). JA can also modulate endocytosis and plasma membrane accumulation of the PIN-FORMED 2 (PIN2) protein (Sun et al. 2011). In addition, JASMONATE INSENSITIVE 1 (MYC2) directly represses PLETHORA (PLT) expression during JAmediated modulation of the root stem cell niche in Arabidopsis and the Arabidopsis P450 protein CYP82C2 modulates JAinduced root growth inhibition (Liu et al. 2010; Chen et al. 2011). Methyl-jasmonate (MeJA)-induced LR formation was also found in rice (Hsu et al. 2013). Jasmonic acid biosynthesis is initiated in the chloroplasts and completed in the peroxisomes. First, 13-hydroperoxy linolenic acid (13-HPLA) is synthesized by the lipoxygenase (LOX), which catalyzes the oxidation of linolenate. 13-HPLA is dehydrated by allene oxide synthase (AOS). The reaction product is cyclized into (9S, 13S)-12-oxo-phytodienoic acid (OPDA) by allene oxide cyclase (AOC). OPDA is further reduced to 3-oxo-2(20 [Z]-pentenyl) cyclopentane-1-octanoic acid (OPC8) by OPDA reductase3 (OPR3). OPC-8:0 CoA Ligase1

Drought is a major environmental stress restricting plant growth and agricultural productivity (Bartels and Sunkar 2005). Altered root architecture is one of the important contributors to drought resistance (Smith and De Smet 2012). Root development has been well studied in the model dicot Arabidopsis thaliana (Peret et al. 2009; De Smet et al. 2012; Petricka et al. 2012; Van Norman et al. 2013). Auxin plays a crucial role in root development. Auxin-dependent signaling pathways are important for lateral root (LR) initiation (Fukaki and Tasaka 2009; Lavenus et al. 2013). In Arabidopsis, auxin causes the degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) to derepress AUXIN RESPONSE FACTOR (ARF) and further activate the expressions of LATERAL ORGAN BOUNDARIES-DOMAIN (LBD) genes which increase LR formation (Okushima et al. 2007; Goh et al. 2012). Other plant hormones regulate root development mainly through the interactions with auxin (Benkova and Hejatko 2009; Fukaki and Tasaka 2009). Jasmonic acid (JA) is usually known as a stress hormone, which has important roles in wound and defense responses of plants. However, it has important functions in plant growth and development as well (Creelman and Mullet 1997; Turner et al. 2002; Browse 2005; Benkova and Hejatko 2009; Wasternack and Hause 2013). JA inhibits primary root (PR) growth and promotes LR formation as well as adventitious root formation (Ahkami et al. 2009; www.jipb.net

Keywords: EDT1/HDG11; jasmonic acid; lateral root; OPCL1; OPR3 Citation: Cai XT, Xu P, Wang Y, Xiang CB (2015) Activated expression of AtEDT1/HDG11 promotes lateral root formation in Arabidopsis mutant edt1 by upregulating jasmonate biosynthesis. J Integr Plant Biol 57: 1017–1030 doi: 10.1111/jipb.12347 Edited by: Jan Traas, University of Lyon, France Received Aug. 25, 2014; Accepted Mar. 2, 2015 Available online on Mar. 4, 2015 at www.wileyonlinelibrary.com/ journal/jipb © 2015 Institute of Botany, Chinese Academy of Sciences

December 2015 | Volume 57 | Issue 12 | 1017–1030

Research Article

School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. *Correspondence: [email protected]

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(OPCL1) can activate JA precursors in the peroxisome. OPC8CoA undergoes three rounds of b-oxidation in the peroxisomes to produce JA (Koo and Howe 2007; Chehab et al. 2011; Knopf et al. 2012; Stenzel et al. 2012). Jasmonic acid can be converted into the bioactive form, JA-Ile, in the cytoplasm to activate JA signaling pathway (Staswick and Tiryaki 2004; Suza and Staswick 2008). JA-Ile can bind to COI1 to cause the degradation of JAZ proteins in the ubiquitin-proteasome pathway (Xie et al. 1998; Xu et al. 2002; Devoto et al. 2005; Lorenzo and Solano 2005). JAZ proteins play their roles as transcriptional repressors. The JAZ downstream transcription factors, such as MYC2, form a complex and precise regulatory network, which can amplify external signals and activate different developmental events in response to different external stimuli (Lorenzo et al. 2004). The edt1 (edt1D) mutant shows improved drought tolerance (Yu et al. 2008). One important contribution is that edt1D has a more extensive root system than the wild type. edt1D is a gain-of-function mutant caused by the strong expression of EDT1/HDG11 in a constitutive fashion (Yu et al. 2008). Overexpression of AtEDT1/HDG11 in rice also conferred enhanced drought tolerance and improved root system (Yu et al. 2013). EDT1/HDG11 encodes a transcription factor belonging to the class IV homeodomain-leucine zipper family, which is a specific gene family of plants (Nakamura et al. 2006; Ariel et al. 2007). It can directly bind to HD (homeodomain) binding sites located in the promoters of downstream target genes. HD binding site is one kind of cis-elements bound by homeodomain-Leucine Zipper protein (Abe et al. 2001; Nakamura et al. 2006). The molecular mechanisms underlying altered root architecture of edt1D were not well understood, although EDT1/HDG11 could downregulate RGAL and IAA28 that might affect edt1D root architecture (Yu et al. 2008). To investigate the molecular mechanisms that underlie the altered root architecture of edt1D, we compared the root transcriptome between the wild type and edt1D and found that the upregulated genes involved in JA biosynthesis or JA signaling pathway were enriched in edt1D root. Further analysis demonstrated that EDT1/HDG11 directly bound to HD binding sites in the promoters of AOS, AOC3, OPR3 and OPCL1 to increase the capacity of JA biosynthesis in the root of edt1D. This is consistent with the elevated JA level observed in edt1D root, leading to more auxin accumulation in the lateral root primordium. Moreover, genetic analysis of edt1D opcl1 (Salk_140659C) double mutant demonstrates that HDG11 is partially dependent on JA in regulating LR formation. Therefore, enhanced JA biosynthesis is one of the mechanisms underlying the EDT1/HDG11-conferred altered root architecture of edt1D.

RESULTS Root transcriptome comparison between the wild type and edt1D To study the molecular mechanisms underlying the altered root architecture of edt1D, root transcriptome of wild type and edt1D were analyzed (Xu et al. 2014). We compared the root transcriptome between the wild type and edt1D seedlings of 3, 6, 10, 15, and 20 d old. In our analysis, the genes were defined as the significantly expressed genes (SEGs) if the change of December 2015 | Volume 57 | Issue 12 | 1017–1030

their transcription levels was greater than or equal to 1.5 folds. It was found that the expression levels of 3717 genes in edt1D root were greater than or equal to 1.5 fold of their expression levels in the wild type root at one time point at least. The LR formation of the wild type was significantly increased at 15 and 20 d old. Moreover, edt1D had significantly more LR formation than the wild type at 15 and 20 day time points (Yu et al. 2008). Therefore, we compared the root transcriptome between the wild type and edt1D of 15- and 20-day-old seedlings. Among the 3717 upregulated genes, only 116 genes were found to be upregulated at 15 and 20 day time points, at which the LR formation of edt1D was significantly increased as compared with the wild type (Table S1) (Yu et al. 2008). Gene Ontology (GO) term enrichment analysis of 116 upregulated SEGs showed that JA-related genes were significantly enriched (Table 1; Figure 1A). To identify the genes that could be potentially regulated by the transcription factor EDT1/HDG11, we searched for HD binding sites in the promoters of 116 candidate genes and found that 72 genes had at least one HD binding site in their promoters (Table S1). Potentially, 62% of the 116 genes could be under direct regulation of EDT1/ HDG11, a significant enrichment over the Arabidopsis genome average 34.4% (Table 2). GO term enrichment analysis of 72 upregulated SEGs showed that JA-related genes are also significantly enriched (Table 3). After cluster analysis using the expression patterns of the 72 upregulated genes, we narrowed down to 26 genes whose expression was progressively increased from day 3 to day 20 for further analysis (Figure 1B). The expression pattern of these genes was consistent with the curve of LR formation (Yu et al. 2008). JA-responsive genes were found as the most enriched functional categories after GO term enrichment analysis of 26 upregulated SEGs (Table 4). Upregulated genes involved in JA biosynthesis and signaling were enriched in edt1D roots Among the 26 upregulated genes, those involved in JA biosynthesis and signaling were significantly enriched (Table 5), including two genes encoding key JA biosynthesis enzymes: AOC3 and OPCL1 (Koo and Howe 2007; Stenzel et al. 2012). In addition, AOS and OPR3 in JA biosynthesis were upregulated in edt1D root at day 20, although they did not appear in the 26 genes sorted out by our standards (Chehab et al. 2011; Knopf et al. 2012). 85.9% of the JA biosynthesis genes contain HD binding site in their promoters (Tables S2, S3). 83.3% of the JA biosynthesis genes were upregulated in edt1D root and could potentially be under direct regulation of EDT1/HDG11, a significant enrichment over the Arabidopsis genome average 34.4% (Table 2). JAZ6 was characterized as a transcriptional repressor in JA signaling pathway and induced by JA (Thines et al. 2007). VEGETATIVE STORAGE PROTEIN 2 (VSP2) was JA-mediated wound marker gene (Wang et al. 2008; Vadassery et al. 2012). ETHYLENE RESPONSE FACTOR 109 (ERF109) was quickly induced as early as 0.5 h after MeJA treatment (Wang et al. 2008). MYB15 and other transcription factors of WRKY family, including WRKY18, WRKY40, WRKY48, and WRKY53, can also respond to JA stimulation (Taki et al. 2005; Zheng et al. 2006; Miao et al. 2007; Wang et al. 2008; Xing et al. 2008). In addition, ASA1, which was very important for JA-induced LR formation and auxin biosynthesis as well as transport, was also upregulated in edt1D root although it did www.jipb.net

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Table 1. Gene Ontology term enrichment analysis of 116 significantly expressed genes (SEGs) in edt1D roots Functional category

abs set

rel set

abs genome

rel genome

P-value

Storage protein Cell rescue, defense and virulence Plant signaling molecules response to JA

5 18

4.31% 15.5%

65 1425

0.22% 5.01%

7.06E-06 1.89E-05

6 At5g24770 VSP2 At3g23250 MYB15 At3g28910 MYB30 At4g31800 WRKY18 At1g80840 WRKY40 At1g76930 EXT4 11 8 (At3g25780 AOC3 At1g20510 OPCL1) 9 (At5g24770 VSP2 At3g23250 MYB15 At3g28910 MYB30 At4g31800 WRKY18 At1g80840 WRKY40 At5g05730 ASA1) 11

5.17%

197

0.69%

1.62E-04

9.48% 6.89%

717 426

2.52% 1.49%

1.75E-04 3.71E-04

7.75%

589

2.07%

7.11E-04

9.48%

841

2.95%

6.70E-04

4

3.44%

117

0.41%

1.37E-03

6 9

5.17% 7.75%

296 692

1.04% 2.43%

1.38E-03 2.16E-03

Electron transport Secondary metabolism (JA biosynthesis) Plant hormonal regulation (JA signaling)

Chemoperception and response Metabolism of phenylpropanoids Response to biotic stimulus Plant specific systemic sensing and response

Gene Ontology term enrichment analysis of 116 SEGs was performed using MIPS website (http://mips.helmholtz-muenchen.de/ proj/funcatDB/). The top 10 enrichment functional categories are shown in the table. abs set, the number of the genes belonged to one functional category in 116 genes; rel set, the percentage of this class of genes in all genes for enrichment analysis; abs genome, the number of the genes belonged to one functional category in the Arabidopsis genome; rel genome, the percentage of this class of genes in all genes of Arabidopsis genome.

not appear in the 26 genes. The significant enrichment of JA signaling components was further confirmed by functional category analysis (Table 4). Other genes that did not have JArelated functions were already reported (Roudier et al. 2002; Gan et al. 2005; Schonrock et al. 2006; Lee et al. 2009; Saga et al. 2012; Schroder et al. 2012; To et al. 2012). The results of root transcriptome comparison showed that the expressions of the genes involved in JA biosynthesis and signaling were significantly increased in the root of edt1D (Tables 1–5, S2). Based on the results of root transcriptome comparison between the wild type and edt1D, the time course expression patterns of the abovementioned genes were further studied in detail. As shown in Figure 2, all JA-related genes were upregulated in edt1D root at 15 and 20 day time points except AOS that was activated only in the root of 20-day-old edt1D seedlings. Our results implicate that these genes involved in JA biosynthesis are candidate targets of EDT1/HDG11. The validation of root transcriptome comparison data To confirm the reliability of root transcriptome comparison data, we examined the expressions of the genes involved in JA biosynthesis or JA signaling pathway in root tissues of 20-dayold wild type and edt1D seedlings by quantitative RT-PCR. The results showed that JA biosynthesis genes (AOS, AOC3, OPR3 and OPCL1) and JA response genes (JAZ6, VSP2, ERF109, ASA1, MYB15, WRKY18, WRKY40, WRKY48 and WRKY53) were www.jipb.net

significantly upregulated in edt1D roots at indicated time points (Figure S1). The results are consistent with root transcriptome comparison data (Figure 2). EDT1/HDG11 directly binds in vivo to the promoter regions of AOS, AOC3, OPR3, and OPCL1 As a transcription factor, EDT1/HDG11 may directly activate the transcription of AOS, AOC3, OPR3 and OPCL1, which all contained HD binding sites in their promoters. To determine whether EDT1/ HDG11 can directly bind to the HD binding sites in the promoters of JA biosynthesis genes in vivo, we performed chromatin immunoprecipitation (ChIP) assay. Promoter sequence analysis identified two HD binding sites in the promoters of AOS, AOC3 and OPCL1, respectively, while only one HD binding site in OPR3 promoter (Figure 3A). All of these HD binding sites have the same sequence (5’-AAATTAAA-3’). The 35S-HA-EDT1/HDG11 transgenic plants were generated and the plants showing similar phenotype to edt1D mutant were used for ChIP assay (Xu et al. 2014). The promoter fragments enriched by anti-HA antibodies (Abmart, www.abmart.com.cn) can be detected by PCR. As shown in Figure 3B, the AOS cis-2, AOC3 cis-1, OPR3 and OPCL1 cis-1 promoter regions spanning the HD binding sites were significantly enriched. These results indicated that EDT1/HDG11 can directly bind in vivo to the HD-binding sites in the promoters of AOS, December 2015 | Volume 57 | Issue 12 | 1017–1030

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Figure 1. Root transcriptome comparison between the wild type and edt1D (A) Jasmonic acid (JA) biosynthesis pathway. The enzyme families involved in JA biosynthesis were marked. (B) Diagram of the gene numbers after different rounds of screen. It was found that the expression levels of 3717 genes in edt1D root were greater than or equal to 1.5 fold of their expression levels in the wild type root. Among the 3717 genes, only 116 genes were upregulated in the roots of both 15 and 20-d-old edt1D seedlings. 72 out of the 116 genes contain HD-binding site in their promoters. Among the 72 genes, only 26 genes showed progressive upregulation from day 3 to day 20.

AOC3, OPR3 and OPCL1 and may transcriptionally regulate these genes in Arabidopsis, which is consistent with the results of root transcriptome comparison and qRT-PCR confirmation for these genes. The ChIP assay was also performed by quantitative RT-PCR (Figure 3C). The result further confirmed the enrichments of AOS, AOC3, OPR3 and OPCL1 promoter fragments by HDG11. HDG11 showed different binding affinities to different sequences belonged to HD binding sites and the sequence 5’-AAATTAAA-3’was the favorite sequence (Xu et al. 2014). We also performed yeast-one-hybrid assay. 30 bp fragments containing HD binding sites were chosen from the promoters of AOS, AOC3, OPR3, and OPCL1. The result of yeast-one-hybrid assay was consistent with the ChIP assay (Figure 3B–D). HDG11 was able to bind to AOS cis2, AOC3 cis1, OPR3 cis, OPCL1 cis1. December 2015 | Volume 57 | Issue 12 | 1017–1030

JA level was elevated in the root of edt1D We previously showed that edt1D had significantly more LRs than the wild type at 10, 12, and 15 d post-germination (Yu et al. 2008), which prompted us to investigate the underlying mechanisms. JA is known to promote LR formation. We measured the endogenous JA levels in the roots of the wild type and edt1D seedlings of 10, 12, and 15 d old when the LR number was significantly different between the wild type (Col-0) and edt1D (Figure 4A). JA content was measured using enzyme-linked immunosorbent assay (ELISA) as described (Yang et al. 2001). The results in Figure 4B show that the roots of 10, 12 and 15-day-old edt1D seedlings contained higher JA levels than the roots of the wild type at the same time points, which is consistent with the phenotype of more LRs of edt1D mutant. www.jipb.net

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Table 2. The ratio of jasmonic acid (JA) biosynthesis genes upregulated in edt1D roots, whose promoter contains at least one HD-binding site Gene family

SEG genes and their numbers

With HD-binding cis-element

JA biosynthesis

12 (At1g55020, At3g45140, At1g17420, At1g72520, At3g22400, At1g73680, At5g42650, At3g25760, At3g25770, At3g25780, At2g06050, At1g20510)

LIPOXYGENASE (LOX) ALPHA-DIOXYGENASE(ALPHA-DOX) AOS AOC

5 (At1g55020, At3g45140, At1g17420, At1g72520, At3g22400) 1 (At1g73680) 1 (At5g42650) 3 (At3g25760, At3g25770, At3g25780)

OPR OPCL1 Arabidopsis genome

1 (At2g06050) 1 (At1g20510) 29110

10 (83.3%) (At1g55020, At1g17420, At3g22400, At1g73680, At5g42650, At3g25760, At3g25770, At3g25780, At2g06050, At1g20510) 3 (60%) (At1g55020, At1g17420, At3g22400) 1 (100%) (At1g73680) 1 (100%) (At5g42650) 3 (100%) (At3g25760, At3g25770, At3g25780) 1 (100%) (At2g06050) 1 (100%) (At1g20510) 10010 (34.4%)

Table 3. Gene Ontology term enrichment analysis of 72 significantly expressed genes (SEGs) containing HD-binding ciselement in their promoters rel set

abs genome

rel genome

P-value

5.55% 12.5% 6.94%

65 717 197

0.22% 2.52% 0.69%

2.28E-05 8.13E-05 1.46E-04

13.8% 16.6%

987 1425

3.47% 5.01%

1.86E-04 2.27E-04

9

12.5%

841

2.95%

2.68E-04

7 (At5g24770 VSP2 At3g23250 MYB15 At3g28910 MYB30 At4g31800 WRKY18 At1g80840 WRKY40 At5g05730 ASA1) 6 (At3g25780 AOC3 At1g20510 OPCL1)

9.72%

589

2.07%

7.30E-04

8.33%

426

1.49%

7.44E-04

5 3

6.94% 4.16%

296 80

1.04% 0.28%

9.39E-04 1.11E-03

Functional category

abs set

Storage protein Electron transport Plant signaling molecules response to JA.

4 9 5 At5g24770 VSP2 At3g23250 MYB15 At3g28910 MYB30 At4g31800 WRKY18 At1g80840 WRKY40 10 12

DNA conformation Cell rescue, defense and virulence Chemoperception and response Plant hormonal regulation (JA signaling)

Secondary metabolism (JA biosynthesis) Response to biotic stimulus Response to wounding

Gene Ontology term enrichment analysis of 72 SEGs was performed using MIPS website. The top 10 enrichment functional categories are shown in the table.

Elevated auxin level in LRPs of edt1D Auxin plays a key role in root development. The auxin level in the root tissues of wild type and edt1D was analyzed. DR5:GFP reporters were introduced into wild type and edt1D by crossing. No difference in green fluorescent protein (GFP) signal was found in the PR tips between wild type and edt1D (Figure 5A). However, significantly more GFP signal was observed in the LRP of edt1D compared with that of wild type (Figure 5B). Gene ontology term enrichment analysis of the 3717 SEGs revealed that the genes involved in auxin biosynthetic process and signaling pathway were also enriched (Table S4). www.jipb.net

HDG11 is partially dependent on JA in regulating LR formation in edt1D To confirm the role of JA in the LR formation of edt1D, we crossed edt1D with opcl1 mutant in which the JA level is reduced and obtained edt1D opcl1 double mutant (Figure 6A, B). The double mutant showed that the LR density of edt1D opcl1 was intermediate between edt1D and opcl1 (Figure 6C), which demonstrates that OPCL1 is one of the targets of HDG11. This result suggests that JA is required for HDG11 to regulate LR formation and other molecular mechanisms may also be involved in the increased LR density of edt1D. December 2015 | Volume 57 | Issue 12 | 1017–1030

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Table 4. Functional distribution of 26 genes selected from the root transcriptome comparison between the wild type and edt1D Functional category

abs set

Plant signaling molecules response to JA

4 At5g24770 VSP2 At3g23250 MYB15 At4g31800 WRKY18 At1g80840 WRKY40 9 5 4 (At5g24770 VSP2 At3g23250 MYB15 At4g31800 WRKY18 At1g80840 WRKY40) 3

Transcriptional control Electron transport Plant hormonal regulation (JA signaling) Response to biotic stimulus

rel set

abs genome

rel genome

P-value

15.3%

197

0.69%

2.97E-05

34.6% 19.2% 15.3%

2117 717 589

7.45% 2.52% 2.07%

6.77E-05 4.27E-04 1.90E-03

11.5%

296

1.04%

2.44E-03

Gene Ontology term enrichment analysis of 26 SEGs was performed using MIPS website. The top five enrichment functional categories are shown in the table.

Table 5. The 26 genes sorted out after three rounds of screen from transcriptome comparison between the wild type and edt1D ID

Locus

Gene name

Function

Reference

1 2 3 4 5 6

At1g20510 At3g25780 At1g72450 At5g24770 At4g34410 At3g23250

OPCL1 AOC3 JAZ6 VSP2 ERF109 MYB15

JA biosynthesis enzyme. JA biosynthesis enzyme. A transcriptional repressor in JA signaling pathway. JA-mediated wound marker gene. JA and CHX induced transcription factor (JCTF). JA induced transcription factor.

7 8

At4g31800 At1g80840

WRKY18 WRKY40

JA and CHX induced transcription factor (JCTF). JA induced transcription factor.

9

At5g49520

WRKY48

10

At4g23810

WRKY53

11

At1g35140

EXL1

12 13

At2g30750 At4g14060

CYP71A12 No

14 15

At4g29780 At4g36430

No No

16 17

At1g11610 At1g16060

CYP71A18 ADAP

18

At1g26400

No

19 20 21 22

At1g66700 At2g33850 At3g08860 At4g11880

PXMT1 No PYD4 AGL14

23 24

At4g22950 At5g44400

AGL19 No

25

At1g26410

No

26

At3g16860

COBL8

Up regulated by MeJA treatment. Stress- and pathogen-induced transcription factor. Response to MeJA treatment. Involved in pathogen defense and leaf senescence. Required for adaptation to carbon (C) - and energy -limiting growth conditions. Response to mechanical stimulus. Camalexin biosynthetic gene. Response to bacterium. Polyketide cyclase/dehydrase and lipid transport superfamily protein. Response to biotic stimulus and defense response. Response to mechanical stimulus and wounding. Peroxidase superfamily protein. Response to other organism and oxidative stress. Camalexin biosynthetic gene. Involved in ABA signaling. Involved in Fatty Acid Biosynthesis. FAD-binding Berberine family protein. Oxidationreduction process SABATH methyltransferase gene Unknown protein Beta-alanine aminotransferase Preferentially expressed in root tissues. N-regulated genes. Involved in vernalization and flowering. FAD-binding Berberine family protein. Oxidationreduction process. FAD-binding Berberine family protein. Oxidationreduction process. Involved in the orientation of cell expansion in the root.

Koo & Howe 2007 Stenzel et al. 2012 Thines et al. 2007 Vadassery et al. 2012 Wang et al. 2008 Taki et al. 2005 Zheng et al. 2006 Wang et al. 2008 Zheng et al. 2006 Wang et al. 2008 Zheng et al. 2006 Xing et al. 2008 Zheng et al. 2006 Miao et al. 2007 Schroder et al. 2012 TAIR

Saga et al. 2012 TAIR TAIR

TAIR TAIR TAIR Lee et al. 2009 To et al. 2012 TAIR TAIR TAIR TAIR Gan et al. 2005 Schonrock et al. 2006 TAIR TAIR Roudier et al. 2002

Functional descriptions of some genes were obtained from TAIR – The Arabidopsis Information Resource (http://www. Arabidopsis.org). December 2015 | Volume 57 | Issue 12 | 1017–1030

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DISCUSSION EDT1/HDG11 upregulates JA biosynthesis to affect root architecture Improved root systems allow plants to absorb more water and nutrients, and enhance their tolerance to drought stress. As we previously reported, the edt1D mutant shows improved drought tolerance and extensive root system with long PR and more LRs (Yu et al. 2008). We continue to unravel the molecular mechanisms that underlie the altered root architecture of edt1D. We compared the root transcriptome between the wild type and edt1D to further study molecular mechanisms of the altered root architecture of edt1D. In order to identify the downstream target genes of EDT1/HDG11, which were involved in root development, especially in the LR formation, we used three rounds of screening (Figure 1B). Twenty-six genes were sorted out after the three rounds of screening (Tables 5, S1). Among the 26 genes, the genes for JA biosynthesis and JA signaling were enriched in the root of edt1D (Tables 4, 5). Consistent with this result is the elevated JA level in edt1D root (Figure 4B). JA can promote LR formation in Arabidopsis (Sun et al. 2009; MorquechoContreras et al. 2010; Raya-Gonzalez et al. 2012). Enhanced JA biosynthesis in the roots of edt1D may cause the phenotype of increased LR formation. Genetic analysis with edt1D opcl1 double mutant also showed that HDG11 was partially dependent on JA in regulating LR formation (Figure 6). It is also known that exogenous JA treatment can inhibit PR growth, but edt1D had improved root system with long PR (Yu et al. 2008; Sun et al. 2009; Raya-Gonzalez et al. 2012). Nevertheless, exogenous JA treatment is different from the elevation of endogenous JA level, because endogenous JA may play its role with strict spatiotemporal constraints. Another possibility is that EDT1/HDG11 may also regulate other downstream signaling pathways that affect PR development. PR phenotype of edt1D may be a result of integration of multiple signaling pathways. It has been reported that HDG11 upregulates cell-wall-loosening protein genes to promote root elongation in Arabidopsis (Xu et al. 2014).

3

Figure 2. Continued. www.jipb.net

Figure 2. The dynamic change of the transcription levels of HDG11 and jasmonic acid (JA)-related genes in root transcriptome of the wild type and edt1D during root development The root transcriptome was compared between the wild type and edt1D seedlings of 3, 6, 10, 15, and 20 d old. The overexpression of EDT1/HDG11 was found in edt1D root. In the 26 genes sorted out by our standards, the transcription levels of JA biosynthesis genes (AOC3 and OPCL1) and JA-related genes (JAZ6, VSP2, ERF109, MYB15, WRKY18, WRKY40, WRKY48 and WRKY53) in edt1D root were greater than or equal to 1.5 folds of their levels in wild type root at 15 and 20 d. The upregulated expressions of JA biosynthesis genes AOS and OPR3 in edt1D root were greater than 1.5 folds at 20 d. In addition, ASA1, which was very important for JA-induced LR formation and auxin biosynthesis as well as transport, was also upregulated in edt1D root, although it did not appear in the 26 genes. DAG, days after germination. December 2015 | Volume 57 | Issue 12 | 1017–1030

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Figure 3. Enrichment of the AOS, AOC3, OPR3 and OPCL1 promoter fragments contained HD binding sites using ChIP-PCR assay (A) The schematic view of the locations of HD binding sites (inverted red triangles) in the promoters of AOS, AOC3, OPR3 and OPCL1 as well as the primers used for PCR (blue lines). (B) ChIP-PCR assay. ChIP assay was conducted using 35S-HA-EDT1/HDG11 transgenic seedlings and anti-HA antibodies (Abmart Shanghai). HA-EDT1/HDG11 was precipitated from input DNA with anti-HA antibodies or with no antibody. The enrichment of DNA fragments was determined by PCR. About 300 bp AOS cis2, AOC3 cis1, OPR3 and OPCL1 cis1 promoter fragments spanning HD binding sites were enriched by anti-HA antibodies. The region of tubulin8 (TUB8) that do not contain HD-box was used as a negative control. (C) Quantitative RT-PCR analysis for ChIP assay. The enrichments of AOS, AOC3, OPR3, and OPCL1 promoter fragments were confirmed by Quantitative RT-PCR. For AOS, AOC3, and OPCL1, the promoter fragments with no enrichment detected in the ChIP-PCR assay were used as negative controls. As only one fragment of OPR3 was analyzed by ChIP-PCR, one new promoter region of OPR3 that do not contain HD binding site was used as negative control in ChIP-Q-PCR. Values are mean  SD (n ¼ 3 experiments, *P < 0.05, ** P < 0.01, *** P < 0.001). Asterisks indicate Student’s t-test significant differences. (D) Yeast-one-hybrid assay for HDG11 binding to the HD binding sites in the promoters of AOS, AOC3, OPR3, and OPCL1. 30 bp promoter fragments contained HD binding sites were chosen for BD vector constructions. A serial yeast dilutions (1:1, 1:10, 1:100 and 1:1000) were grown on SD medium lacking Leu, and Trp (-Leu-Trp) and SD medium lacking Leu, Trp and His (-His-Leu-Trp), respectively. The empty AD (pGADT7) and BD (pHIS2) vectors were used for negative controls.

We selected the genes based on the fact that they were upregulated at least 15 and 20 d post-germination (Figure 1B). However, root architecture and JA levels are changed as early as 10 d post-germination (Figure 4). We found the significant enrichment of JA biosynthesis and signal gene through the analysis of the upregulated genes at two time points (15 d December 2015 | Volume 57 | Issue 12 | 1017–1030

and 20 d). We also found that the expression of two JA biosynthesis genes, AOC3 and OPCL1, in the root of edt1D, was upregulated as early as 10 d post-germination, which could cause the changed JA levels and root architecture (Figures 2, 4). In addition, other molecular mechanisms may also exist in the root architecture regulation of edt1D. www.jipb.net

AtHDG11 promotes Arabidopsis lateral root formation

Figure 4. Elevated levels of endogenous jasmonic acid (JA) in edt1D roots (A) The density of lateral roots (LRs) of the wild type and edt1D seedlings grown on MS were counted at the indicated time points. Values are mean  SD (n ¼ 12 seedlings, *P < 0.05). Asterisks indicate Student’s t-test significant differences. The assay was repeated three times and the same result was obtained. (B) JA contents were measured by enzyme linked immunosorbent assay (ELISA) using root tissues of 10, 12 and 15-d-old wild type and edt1D seedlings. Values are mean  SD (n ¼ 3 experiments, *P < 0.05, **P < 0.01). Asterisks indicate Student’s t-test significant differences.

We noticed that the expressions of AOC3, AOS, OPR3 and OPCL1 in the roots of wild type at 15 d also appear slightly elevated (Figure 2). JA signaling pathway may be involved in the process of LR formation of wild type under normal conditions. This pathway is enhanced in the roots of edt1D by the overexpression of HDG11, which causes the increased LR number of edt1D as compared with the wild type (Figures 2, 4A). HDG11 levels are much higher at 3 d after germination already, while activation lag was found in the expressions of JA biosynthesis genes, as downstream target genes of HDG11 (Figure 2). The repressive regulatory mechanism may exist in root tissues of both wild type and edt1D, which limits the role of JA signaling pathway to specific developmental stage. www.jipb.net

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The molecular mechanisms underlying the altered root architecture of edt1D The transcription levels of the JA-related genes were upregulated by the overexpression of EDT1/HDG11 in edt1D root (Figures 2, S1). Our result showed that EDT1/HDG11 can directly bind to the HD binding sites in the promoters of JA biosynthesis genes AOS, AOC3, OPR3 and OPCL1 to elevate their transcript levels, subsequently leading to increased JA level in roots (Figures 3, 4B). AOS, AOC3, OPR3, and OPCL1 encode the key enzymes of the JA biosynthesis pathway (Stintzi and Browse 2000; Von Malek et al. 2002; Koo et al. 2006; Stenzel et al. 2012). The main expression of AOS was found in leaves and floral organs, suggesting its important role for floral organ abscission and pollen maturation (Kubigsteltig et al. 1999). AOS could be induced by jasmonates in Arabidopsis (Laudert and Weiler 1998). The activated expression of AOS was observed in root tips and LRs under jasmonate treatment, although the AOS expression level is low in root tissues under normal conditions (Kubigsteltig et al. 1999). Our study reveals that overexpression of HDG11 promotes the transcription of AOS in the root tissues (Figures 2, 3). HDG11-enhanced expression of AOS suggested that HDG11 may be responsible for activation of the AOS potential function in LR formation. There are four members (AOC1, AOC2, AOC3 and AOC4) of the AOC gene family with organ-specific expression pattern (Stenzel et al. 2012). The expression of AOC3 was observed in meristematic cells of PR and the sites of LR formation (Stenzel et al. 2012). In addition, the expression of OPR3 was detected in the vascular bundles of roots and the base of LRs (Li et al. 2013). The weak expression of OPCL1 was found in the vascular system of roots under normal conditions and high expression of OPCL1 could be found in the zone of cell division proximal to the root tip, vascular system and primordial cells of LRs under MeJA treatment (Kienow et al. 2008). The expression pattern of AOS, AOC3, OPR3, OPCL1 suggests that they may play potential important roles in LR formation. The expression of JA-related targets was not elevated in the wild type as sharply as in edt1D (Figures 2, S1). The expression of these JA biosynthesis genes are enhanced mainly in the sites of LR formation, which is also consistent with the expression of HDG11 in the sites of LRPs (Nakamura et al. 2006). As downstream target of HDG11 (Figure 3), AOS, AOC3, OPR3, OPCL1 are activated by HDG11, resulting in the enhanced JA biosynthesis at the sites of LR formation. How could EDT1/HDG11-enhanced JA biosynthesis affect root architecture? Based on our results, ASA1, which is very important for JA-induced LR formation and auxin biosynthesis as well as transport (Sun et al. 2009), was upregulated in edt1D root (Figures 2, S1). We also found that edt1D had more signal of DR5:GFP in LRPs than wild type (Figure 5). The elevated JA level in the root tissue of edt1D may activate ASA1-dependent auxin biosynthesis in the sites of LR initiation to promoter LR formation. In contrast, IAA28, as a transcription repressor of auxin signaling pathway, showed reduced expression in edt1D root. These data suggest that EDT1/HDG11 may regulate root architecture through the crosstalk between JA and auxin. Why increased JA biosynthesis enhances DR5:GFP expression in the primordium of LR, but not in the tip of PR (Figure 5)? The overexpression of HDG11 increases the content of JA in edt1D root (Figure 4B). First of all, we found that HDG11-regulated JA December 2015 | Volume 57 | Issue 12 | 1017–1030

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Figure 5. Fluorescence observation of DR5-GFP in the Col-0 and edt1D background (A, B) The comparison of DR5-GFP expression levels between 20-d-old wild type and edt1D. DR5-GFP levels in the primary root (PR) tips (A) and lateral root primordia (LRPs) (B) of 20-d-old Col-0 and edt1D seedlings were shown. Three independent lines were analyzed for Col-0 and edt1D background respectively and same result was obtained. Bar ¼ 20 mM. Green fluorescent protein (GFP) fluorescence was quantified using ZEISS710 program on confocal sections under the same microscope settings. Values are mean  SD (n ¼ 15 images, ***P < 0.001). Asterisks indicate Student’s t-test significant differences.

biosynthesis genes (AOS, AOC3, OPR3 and OPCL1) are mainly expressed in the site of LR formation and stele, although the expression of AOS, which is low in root tissues under normal conditions, was observed in root tips under jasmonate treatment (Kubigsteltig et al. 1999; Kienow et al. 2008; Stenzel et al. 2012; Li et al. 2013). Therefore, HDG11 may enhance JA biosynthesis mainly in the site of LR rather than PR tip. In addition, JA may interact with auxin to promote LR formation. The regulatory factors mediated the crosstalk of JA and auxin may not be expressed in PR tip, so auxin accumulation may not be found even if JA biosynthesis is enhanced in PR tip of edt1D. The unchanged auxin level in PR tip of edt1D is in consistent with the phenotype of edt1D, as HDG11-promoted root elongation is due to the upregulated December 2015 | Volume 57 | Issue 12 | 1017–1030

expressions of cell-wall-loosening protein genes (Xu et al. 2014). It is known that EDT1/HDG11 downregulates RGAL, encoding a RGA-like DELLA protein, as a repressor of GA signaling, and IAA28, repressing LR formation, which may contribute to the improved root system of the edt1D with more elongated PR and increased LR formation (Rogg et al. 2001; Wen and Chang 2002; Fu and Harberd 2003; Achard et al. 2006; Yu et al. 2008). In addition, EDT1/HDG11 may also regulate other signaling pathways to improve root architecture based on the root transcriptome comparison between the wild type and edt1D. Further study is required to elucidate the whole regulatory network of EDT1/HDG11. www.jipb.net

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Figure 6. OPCL1 acts downstream of HDG11 (A) Jasmonic acid (JA) contents were measured by enzyme linked immunosorbent assay (ELISA) using 12-d-old wild type and opcl1 seedlings. Values are mean  SD (n ¼ 3 experiments,  P < 0.01). Asterisks indicate Student’s t-test significant differences. (B) The expressions of OPCL1 in both wild type and edt1D opcl1 were detected by RT-PCR. The double mutant edt1D opcl1 was screened by glufosinate firstly and then they were identified by RT-PCR. RNA was extracted from rosette leaves of wild type and edt1D opcl1 adult plants. TUB8 was used as a loading control. (C) The density of lateral roots (LRs) of Col-0, edt1D, edt1D opcl1 and opcl1 seedlings grown on MS were counted at the indicated time points. Values are mean  SE (n ¼ 20 seedlings,  P < 0.05,  P < 0.01,  P < 0.001). Asterisks indicate Student’s t-test significant differences.

In conclusion, these results provided a possible mechanism for the increased LR density of edt1D. We found that the homeodomain transcription factor EDT1/HDG11 directly binds to HD binding sites in the promoters of AOS, AOC3, OPR3 and OPCL1 to upregulate their expression levels and increase the content of JA in the root. JA is known to induce the expressions of JA biosynthesis genes (Kazan and Manners 2008). The increased content of JA in the root could further induce its own biosynthesis through positive feedback. Then,

JA could activate the downstream cascade signal transduction. JA response genes, included transcription factors, could be activated to regulate the process of plant development and the response to the external environment. In the root of edt1D, JA may interact with auxin to promote LR formation (Figure 7).

MATERIALS AND METHODS Plant materials and growth conditions The wild type used in our study was Arabidopsis thaliana ecotype Columbia-0 (Col-0). After surface sterilization for 10 min in 10% bleach, Arabidopsis seeds were washed five times at least with sterile water and kept at 4 °C for 2 d in darkness for vernalization. Then, seeds were germinated on Murashige and Skoog (MS) solid medium at 22°C under 16-h light/8-h dark cycles.

Figure 7. The regulatory network of HDG11 underlying the increased lateral root (LR) formation of edt1D HDG11 directly binds to HD binding sites in the promoters of AOS, AOC3, OPCL1 and OPR3 to upregulate their transcript levels and increase JA content, which may activate auxin signaling pathway to increase LR formation. www.jipb.net

Microarrays The seeds were germinated on MS solid medium with or without 50 mg L1 of glufosinate ammonium horizontally. Then, the seedlings were transferred to new MS solid medium and grown vertically. After 3, 6, 10, 15, and 20 day, RNA was extracted from the root tissues of edt1D and the wild type using TRIzol reagent (Invitrogen, Carlsbad, California, USA). An Affymetrix gene chip was carried out and analyzed by Capitolbio Corporation (Beijing, China). GO analysis was performed on the MIPS website (http://mips. helmholtz-muenchen.de/proj/funcatDB/) (Xu et al. 2014) and DAVID Bioinformatics Resources (Huang da et al. 2009a, b). The lower the P-value, the higher the degree of enrichment. December 2015 | Volume 57 | Issue 12 | 1017–1030

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JA quantification Seedlings of the wild type and edt1D were grown on MS medium vertically. Then, the root tissues of these seedlings were collected at 10 day, 12 day and 15 day time points. JA contents were examined by ELISA (Yang et al. 2001). The results were detected by ELIASA (ELX800, BIO-TEK). Gene expression analysis Total RNA was isolated from root tissues by the TRIZOL reagent (Invitrogen). Prime Script RT reagent Kit (TaKaRa, Japan) was used for the reverse transcription reaction. The quantitative RT-PCR was carried out on a Step One Real-Time PCR system (Applied Biosystems, Carlsbad, California, USA) with the SYBR green (SYBR Premix Ex Taq II, TaKaRa) using the fast program. The conditions were 95°C for 30 s and 40 cycles of 95 °C, 2 s, 60°C, 30 s, followed a melting curve analysis. The transcription levels of target genes were detected by specific primers and the primer pairs were listed in Table S5. The UBIQUITIN5 (UBQ5) was used as an internal control to examine the relative expression levels of target genes. The search for HD binding site The HD binding sites in the promoters were searched using AtcisDB – Arabidopsis cis-regulatory element database (http:// Arabidopsis.med.ohio-state. edu /AtcisDB/). The 3000 bp promoter sequences of JA biosynthesis genes were obtained from TAIR – the Arabidopsis information resource (http:// www.Arabidopsis.org). HD binding sites with different types of sequences were searched in the promoter of each gene to confirm the results of AtcisDB subsequently. ChIP-PCR assay ChIP was performed as previously described using 10-dold 35S-HA-EDT1/HDG11 transgenic seedlings and anti-HA antibodies (Abmart, Shanghai, China) (Gendrel et al. 2005). HA-EDT1/HDG11 was precipitated from input DNA with anti-HA antibodies or with no antibody. The input DNA and purified DNA were used as templates. Specific primer pairs, which can amplify approximate 300 bp fragments, were used for the detection of the chromosome regions enriched by EDT1/HDG11. The primer pairs were listed in Table S5. The TUBULIN BETA 8 (TUB8) was used as a negative control. PCR conditions were 95°C for 3 min, 40 cycles of 95 °C , 30 s, 60 °C, 30 s and 72 °C, 30 s, and 72 °C for 10 min. Yeast one-hybrid assay The yeast one-hybrid assay was performed as described previously (Xu et al. 2014). Confocal microscopy analysis The fluorescence of GFP in the root tissues of plants was observed as described previously using a ZEISS710 confocal laser scanning microscope (Wang et al. 2014). Fluorescence intensity was quantified with the ZEISS710 program on confocal sections acquired with the same microscope settings. Accession number Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases December 2015 | Volume 57 | Issue 12 | 1017–1030

under the following accession numbers: EDT1/HDG11, At1g73360; AOS, At5g42650; AOC3, At3g25780; OPR3, At2g06050; OPCL1, At1g20510; JAZ6, At1g72450; VSP2, At5g24770; ERF109, At4g34410; MYB15, At3g23250; WRKY18, At4g31800; WRKY40, At1g80840; WRKY48, At5g49520; WRKY53, At4g23810; ASA1, At5g05730; UBQ5, At3g62250; TUB8, At5g23860.

ACKNOWLEDGEMENTS This work was supported by grants from the Ministry of Science and Technology of China (MOST, 2012CB114304, 2011ZX08005-004), the Chinese Academy of Science (CAS, KSCX3-YW-N-007), and the National Nature Science Foundation of China (NNSFC, 30830075, 90917004). The authors thank the Arabidopsis Biological Resource Center for providing the seeds of opcl1 (Salk_140659C).

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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site. Figure S1. Confirmation of the transcriptome comparison data for the selected genes by quantitative RT-PCR The root tissues of 20-day-old wild type and edt1D seedlings were used. Total RNA was isolated from root tissues as indicated by the TRIZOL. Then, quantitative RT-PCR was carried out. The transcription levels of target genes were examined using specific primers which were listed in Table S5. The ubiquitin5 transcription level was used as an internal control to examine the relative transcription levels of the tested target genes. Values are mean  SD (n ¼ 3 experiments,  P < 0.05,  P < 0.01,  P < 0.001). Asterisks indicate Student’s t test significant differences. All above-mentioned JA-related genes were upregulated in edt1D root at 20 day, which was consistent with root transcriptome comparison data. Table S1. The 116 genes sorted out after 2 rounds of screen from root transcriptome comparison analysis between the wild type and edt1D and the HD-binding sites in their promoters Table S2. The ratio of upregulated genes with HD-binding site in different functional categories Table S3. HD binding sites in the promoters of JA biosynthesis genes Table S4. The enrichment of genes involved in auxin biosynthetic process and signaling pathway Table S5. Primers used in this study

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