Fig 1.
Transcriptomic analysis of class 1 XTH genes during lateral root (LR) initiation.
(A) After gravitropic stimulation to induce the synchronized initiation of lateral root primordia (LRP) at the surface of bending roots, LRP stages from I to VIII, in accordance with previous reports, were assessed every 6 hours for 0 to 54 hours after gravity stimulation and are represented here as a percentage of the total number of observed LRP at each time point. At least 70–80 LRP were observed at each time point. (B, C) All class 1 XTH gene expression patterns at each time point during LR initiation were measured every 6 hours from 6 to 54 hours pgi. Bending roots of a population of 5-day-old seedlings were microdissected at 10 time points, which were then used for RNA extraction (approximately 220 per time point for three independent replicates). Expression patterns of all class 1 XTH genes are shown. The error bars denote SDs (n = 4). Student’s t-test was applied. The asterisks (*) show statistically significant differences (p<0.05).
Fig 2.
XTH9 gene mutation affects lateral root (LR) development.
(A) The genomic organization of the XTH9 gene. The white boxes show the positions and sizes of the XTH9 gene exons. The black box indicates the structure of the T-DNA, the site of which is indicated by the triangle. The genomic sequences used to complement the xth9 mutation are underlined with a thick line. (B) XTH9 transcript levels in wild-type (WT) (Col-0) plants, the xth9 mutant, and xth9 complementary lines. The mRNA abundance of the XTH9 gene in the roots was measured using RT-PCR in various genotype backgrounds of Arabidopsis. (C) Root phenotypes of WT plants, the xth9 mutant, and complementary lines. The plants were grown vertically on media for 10 days. The white bar indicates 1 cm. (D) Primary root length and (E) total LR length. (F) LR density analysis of WT plants, the xth9 mutant, and xth9 complementary lines. The error bars denote SDs (n = 16–21). (G) Phenotypic analysis of lateral root emergence (LRE) was achieved by synchronizing LR formation with a gravity stimulus for 18 and 42 hours. Compared with the WT plants, the xth9 mutants showed delayed LRE. Mutant plants transformed with a 4.9-kb-long full-length XTH9 genomic fragment exhibited a WT LRE phenotype for LR induction. The data are shown as percentages, and the error bars represent SDs (n = 15–17). At least 80–100 total LR primordia were observed for each plant, and the asterisks (*) indicate statistically significant differences (p<0.05).
Fig 3.
Observation of proXTH9::GUS transgenic lines during lateral root (LR) development.
(A–H) GUS staining in proXTH9::GUS Arabidopsis roots at various developmental stages. The seedlings were incubated on media supplemented without (I, J) or with (K, L) 5 mM nitrate for 6 hours. Images of the leaves (I, K) and LRs (J, L) were obtained, and representative images are shown. (M) Relative GUS activity before and after nitrate treatment is shown. Leaf GUS activity in the absence of nitrate was used as a control. The asterisks (*) show statistically significant differences (p<0.05) and the error bars represent SDs (n = 4).
Fig 4.
XTH9 gene mutation altered lateral root (LR) development in response to nitrate treatments.
(A–F) Effects of various concentrations of nitrate (10, 50, 100, 500, 1000, 5000, and 10000 μM) on root growth. WT, xth9 mutant, and the complementary line 1 were grown vertically in media supplemented with various nitrate concentrations for 8 days. (G) Primary root length; (H) LR density; (I) Lateral root length were quantified. LR density was calculated by dividing the LR number per 1 cm of primary root. The error bars represent SDs (n = 22–31). Bars with different capital letters indicate significant differences among different concentrations of nitrate treatment in the given genotype by ANOVA analysis. The white bars indicate 1.2 cm.
Fig 5.
Overexpression of the XTH9 gene increased lateral root (LR) development and improved low-nitrate tolerance.
(A) Root phenotype observation and analysis of (B) LR density, (C) total LR length, and (D) primary root length in WT and XTH9 overexpression lines. Bars with different capital letters indicate significant differences among different nitrate treatment in the given genotype by ANOVA analysis. The white bar indicates 1 cm. (E) Phenotypic analysis of lateral root emergence (LRE) was achieved by synchronizing LR formation with a gravity stimulus for 15 and 36 hours. Compared with the WT (Col-0) plants, the XTH9 overexpression plants showed increased LRE. The data are shown as percentages, and the error bars represent SDs (n = 19–22). The error bars denote SDs (n = 16–23). Approximately 90 total LRP were observed in each plant. (F) WT and transgenic plants were grown vertically in media supplemented with 10 μM nitrate for 8 days. (G) Average LR lengths were quantified. The error bars represent SDs (n = 20–30). Bars with different capital letters indicate significant differences among different concentrations of nitrate treatment in the given genotype by ANOVA analysis. The white bars indicate 1.2 cm.
Fig 6.
Auxin regulates XTH9 expression in an ARF7/19-dependent manner.
(A) XTH9 expression level in the arf7arf19 double mutant background. RNA was isolated from 7-day-old Arabidopsis roots. (B) After treatment with 1 mM IAA for the indicated time, the wild-type (WT) and arf7arf19 double mutant roots were collected at the indicated time points. Values were determined from three independent plant roots. The auxin-dependent repression of XTH9 expression in the roots is ARF7/19 dependent. (C) Relative length of lateral root per 1 cm of primary root and (D) XTH9 expression levels in the WT, arf7arf19-1 and arf7arf19-1 & 35S::XTH9 transgenic plants. *indicates significant differences (p<0.05). The error bars represent SDs (n = 3).
Fig 7.
The nitrate response of XTH9 is altered in afb3-1, slr-1 mutant background and XTH9 is still regulated by nitrate in NR-null nia1nia2 mutant.
(A) WT (Col-0) and afb3-1 and slr-1 mutant plants were grown on media supplemented with ammonium succinate for one week and subsequently treated with 6 mM KNO3 or 6 mM KCl for 1–3 hours. The XTH9 gene expression level in the roots was measured via RT-qPCR. (B) Nitrate reductase-null plants (nia1nia2) were grown in media supplemented with ammonium succinate for one week and then treated with 6 mM KNO3 or 6 mM KCl for 1–3 hours. The RNA level of the XTH9 gene was measured via RT-qPCR. KCl treatment results are shown with white bars, and the KNO3 treatment results are shown with black bars. *indicates significant differences (p<0.05). The error bars represent SDs (n = 18–24).
Fig 8.
OBP4 regulates plant root system architecture.
(A) Phenotypic observation and (B) analysis of the root system in constitutive overexpression and knockdown OBP4 transgenic plants. The white bar indicates 1.2 cm in length. (C) Phenotypic analysis of lateral root emergence (LRE) was performed by synchronizing lateral root (LR) formation with a gravity stimulus for 22 and 38 hours. Compared with that in the control plants, the LRE in the OBP4 overexpression plants and OBP4 knockdown plants was delayed and promoted, respectively. The data are shown as percentages and the error bars represent SDs (n = 8–12). Approximately 24–36 lateral root primordia (LRP) were observed in 35S::OBP4 plants. Approximately 100 LRP were observed in OBP4 knockdown transgenic plant. *indicates significant differences (p<0.05).
Fig 9.
Relative expression of XTH9 and OBP4 genes in response to nitrate treatments and OBP4-XTH9 in control of LR growth in response to variation of nitrate concentrations.
Wild-type (WT) (Col-0) plants were grown on media supplemented with ammonium succinate for one week and subsequently treated with various concentrations of nitrate for 2 hours, and the relative (A) XTH9 and (B) OBP4 gene expression levels were assessed. (C) Phenotypic observations and analysis of (D) the primary roots, (E) the lateral root (LR) density, (F) the LR length of 4-day-old vector control, OBP4-RNAi, OBP4-RNAi-1 & 35S::XTH9-1 and OBP4-RNAi-1 & xth9 transgenic plants in response to various concentrations of nitrate (50, 100, 200, 1000, 5000, 8000, and 10000 μM) treatment for five days. LR density was calculated by dividing the LR number per 1 cm of primary root. *indicates significant differences (p<0.05). The error bars show SDs (n = 20–32). The white bars indicate 1.2 cm.
Fig 10.
The OBP4-XTH9 module regulates lateral root (LR) development in response to nitrate signaling.
Temporal control of the lateral root emergence (LRE) gene regulatory network involving OBP4 and XTH9. Auxin is perceived by AFB3 and triggers the degradation of IAA14, which releases ARF7/19. Auxin regulates XTH9 expression in an ARF7/19-dependent manner. XTH9 is also negatively regulated by OBP4, which fine-tunes the XTH9 expression level in response to environmental nitrate availability. This synergistic regulation of OBP4-XTH9 has a specific function in lateral root (LR) development in response to nitrate signaling in Arabidopsis.