Fig 1.
PRH1 is a downstream target of ARF7-mediated auxin signaling.
(A) Eight-day-old seedlings of WT and arf7. Scale bar: 1 cm. (B) The ratio between the number of the emerged lateral roots and the length of the primary root of WT and arf7 seedlings. Data shown as mean±SE, three biological replicates in the experiment, 20 plant seedlings for each repeat. ***: the mutant’s performance differs significantly (P<0.001) from that of WT. (C) Venn diagram indicating the overlap of differentially expressed genens between the four datasets (Col-NAA VS Col-Mock, arf7-NAA VS arf7-Mock, arf7-Mock VS Col-Mock and arf7-NAA VS Col-NAA) based on RNA-seq. RNA-seq analysis: RNA was extracted from the primary roots of eight-day-old seedlings exposed or not exposed to 10 μM NAA for 4 hours. NAA: naphthalene acetic acid. (D) Heat map of the differentially expressed genes induced by auxin and regulated by ARF7 according to RNA-seq data. (E) The relative transcript abundance of PRH1 in the roots of eight-day-old WT and arf7 seedlings exposed (green) or not exposed (orange) to 10 μM NAA for 4 hours. The relative transcript abundance is relative to the untreated WT. Values represent averages of three biological replicates in the experiment, and the total mRNA was extracted from about 100 seedlings for each repeat. Error bars represent SE. Different letters atop the columns indicate significant (P<0.001) differences in abundance.
Fig 2.
Histological localization and expression of PRH1pro:GUS transgene during LR formation.
(A) and (B) PRH1 is expressed in the cells overlying/surrounding the lateral root primordium(LRP) in WT seedlings not exposed to auxin(A), and the intensity of its expression is strongly enhanced when the plants were treated with 10 μM naphthalene acetic acid (NAA) for 4 hours (B). (C) and (D) GUS activity is suppressed in non-treated arf7 seedlings (C), and even more so in treated ones (D). NAA: naphthalene acetic acid. Before GUS staining the seedlings were soaked for 4 hours in liquid half strength Murashige and Skoog (MS) medium, which containing 10 μM NAA or not. The developmental stages (I through VII and E) of the LRs are indicated in the top left corner. GUS (β-glucuronidase) signals are shown in blue. Stages I to VII of primordia were based on the classification by Malamy and Benfey [1], E: Emerged lateral roots, Scale bars: 50 μm.
Fig 3.
PRH1 acts downstream of ARF7-mediated lateral root (LR) development.
(A) Density of primordia at given stages. Stages I to VII of primordia were based on the classification by Malamy and Benfey [1], E: emerged lateral roots, *,**, ***: means differ significantly (P<0.05, P<0.01, P<0.001) from the WT control. (B) The synchronized initiation of lateral root primordia were induced using gravitropic stimulation at the site of root bending in the 3-day-old seedlings of WT, prh1-1, prh1-2 and prh1-3. Phenotypic analysis of lateral root (LR) emergence was achieved after 18 h and 42 h gravistimulus compared with WT (Col-0) and the LR emergence is delayed in prh1 mutants. Lateral root primordium stages (from I to VII according to previous descriptions from 1997 Benfey [1]) were analyzed from 30 seedlings. (C) Eight-day-old seedlings of WT, arf7, prh1-1 mutants and transgenic lines harboring either 35S::PRH1 or 35S::PRH1/arf7. The upper panel shows the seedlings grown on 1/2 MS medium without NAA addition and the lower panel shows the seedlings grown on 1/2 MS medium containing 30 nM NAA. Bar: 1 cm. (D and E) Density of lateral root primordia (D) and emerged lateral roots (E) of the 8-d-old seedlings grown on the half strength Murashige and Skoog (MS) medium containing 30 nM NAA or not. The percentage values represent the increase with NAA treatment. The density was the ratio between lateral roots number and the length of primary root. NAA: naphthalene acetic acid. Different letters atop the columns indicate significant (P<0.05) differences in abundance. Data shown as means±SE, three biological replicates in the experiment, 20 plant seedlings for each repeat.
Fig 4.
PRH1 is regulated by ARF7 at the transcriptional level.
(A) Structure of PRH1 promoter and the fragments used in the CHIP-qPCR assay. AuxREs are indicated by red squares, and black lines show the promoter regions containing the AuxREs used in this assay. NC: negative control. AuxREs: auxin response elements. (B) ARF7 transactivates the PRH1 promoter in A. thaliana leaf protoplasts. The left hand panel is a schematic of the effector (35S::ARF7) and reporter (PRH1pro:LUC) constructs. The empty vector pBI221 was used as a negative control; the right hand panel shows the ratio of ARF7 drived LUC and the empty vector (negative control) to 35S promoter drived REN respectively. LUC: firefly luciferase activity, REN: renilla luciferase activity. Values shown as means±SE, three biological replicates in the experiment. *: means differ significantly (P<0.05) from the negative control. (C) ARF7 is associated with the PRH1 promoter according to a CHIP-qPCR assay. Chromatin isolated from a plant harboring 35S::MYC-ARF7 and a WT mock control was immunoprecipitated with anti-MYC antibody following the amplification of regions P1, P2 and P3. The coding region segment NC was used as the negative control. The ChIP signal represents the ratio of bound promoter fragments (P1-P3) after immunoprecipitation to total input without immunoprecipitation. Values shown as means±SE, three biological replicates in the experiment. **, ***: means differ significantly (P<0.01, P<0.001) from the WT control. (D) Physical interaction of ARF7 with the PRH1 promoter according to a Y1H assay. The plasmid pGADT7-ARF7 was introduced into Y1H Gold cells harboring the reporter gene PRH1pro:AbAr and the cells were grown on SD/-Ura-Leu medium in the presence of 30 or 50 ng/mL aureobasidin A (AbA). The empty vector pGADT7 was used as a negative control.
Fig 5.
The transcription of PRH1 is activated by LBD.
(A) LBDs associated with the promoter of PRH1 according to a CHIP-qPCR assay. The upper panel shows the structure of the PRH1 promoter, and five uniformly distributed sites (black lines) were selected for the CHIP-qPCR assay. Sites II and III include the LBD29 and LBD18 binding motif (See S6 Fig). NC: negative control. The red lines indicate the AuxREs (auxin response elements) and the green lines indicate LBD-binding elements [12, 44]. The lower panel demonstrates the ratio of bound promoter fragments (Ⅰ-Ⅴ) after immunoprecipitation to total input without immunoprecipitation. The samples were derived from WT seedlings (negative control) and those harboring one of the transgenes 35S::Myc-LBD16 (Myc-LBD16), 35S::Myc-LBD18 (Myc-LBD18), Super::LBD29-GFP (LBD29-GFP) [44, 49]. Values shown as means±SE, three biological replicates in the experiment. * Represents a comparison of the signal arising from the Myc/GFP-fused LBD with WT in the corresponding genomic region. *, **: means differ significantly (P<0.05, P<0.01) from the WT control. (B) LBDs transactivate the PRH1 promoter in A. thaliana leaf protoplasts as shown by a transient dual-luciferase assay. The reporter gene (PRH1 promoter driving LUC) was co-transformed with one or two of the constructs 35S::LBD16, 35S::LBD18 and 35S::LBD29. The empty vector pBI221 was used as negative control. Values shown as means±SE, three biological replicates in the experiment. *, **: means differ significantly (P<0.05, P<0.01) from the WT control.
Fig 6.
The over-expression of PRH1 partially alleviates the defective development of lateral roots (LRs) in the lbd mutant.
(A) Eight-day-old seedlings of WT, the mutants lbd16, lbd18 and lbd29, and the transgenic lines harboring 35S::PRH1. The upper panel shows the seedlings grown on 1/2 MS medium without NAA addition and the lower panel shows the seedlings grown on 1/2 MS medium containing 30 nM NAA. Scale bar: 1 cm. (B and C) Density of lateral root primordia (B) and emerged lateral roots (C) in eight-day-old seedlings grown on the 1/2 MS medium containing 30 nM NAA or not. The percentage values represent the increase with NAA treatment. The density was the ratio between lateral roots number and the length of primary root. NAA: naphthalene acetic acid. Data shown as means±SE, three biological replicates in the experiment, 20 plant seedlings for each repeat. Different letters atop the columns indicate significant (P<0.05) differences in abundance.
Fig 7.
A working model of the regulation of LR development by PRH1.
Auxin induced PRH1 expression is dependent on both ARF7 and LBDs. LAX3, which acts downstream of LBD29, also indirectly regulates PRH1 expression through controlling IAA accumulation in the epidermits and cortex. PRH1 may promote LR emergence by regulating the expression of EXPANSIN genes. ARF7 is expressed in pericycle and primordium [33]; LBD16 is expressed in the primordium at earlier stages [9]; LBD18 and LBD29 are expressed in the LR primordium and overlying tissues [27]; LAX3 is expressed in the epidermis and cortex cells above the primordia [27]. The histological localization of PRH1 is in the cells overlying/surrounding the lateral root primordium. PRH1 might regulate LR emergence through influencing the expression of EXPs that affecting cell wall loosening.