Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation

Abstract

Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide's mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF's mechanism of action could be to interfere with the BR signalling pathway.

Keywords: Root development; brassinolide; peptide hormone..

Fig. 1.

AtRALF1 gene silencing in transgenic lines (irAtRALF1). (A) Transcript levels of the AtRALF1 gene in transgenic plants. Transcript levels were examined by RT-PCR using RNA samples extracted from 10-d-old roots of plants grown on half-strength MS agar plates. GAPDH was used as an internal control. WT, wild type. (B) Root length of AtRALF1-overexpressing (35S:AtRALF1, black columns) and AtRALF1-silenced (irAtRALF1, grey columns) transgenic lines. (C) Hypocotyl length of etiolated transgenic plants. (D) Number of emerged lateral roots per cm in transgenic plants. Root length and number of emerged lateral roots were measured and counted in 5- and 10-d-old seedlings respectively (n>30). Hypocotyl length was measured in 5-d-old etiolated plants. Error bars indicate standard deviation (SD). Columns with the same letter are not significantly different (P<0.01). All experiments were repeated at least three times (independent biological replicates).

Fig. 2.

Root and hypocotyl cell length of AtRALF1-overexpressing (35S:AtRALF1) and AtRALF1-silenced (irAtRALF1) transgenic lines. (A) Root cell length of 35S:AtRALF1 plants (black columns) and irAtRALF1 (grey columns) plants. Panels below the graph are confocal images of the root differentiation zone. Cells from root endodermis of 10-d-old seedlings were measured (n=30 cells per root). WT, wild type. (B) Hypocotyl cell length of 35S:AtRALF1 (black columns) and irAtRALF1 (grey columns) plants. Panels below the graph are confocal images of the base of hypocotyls. Epidermal cells from hypocotyls of 10-d-old seedlings were measured (n=6 cells per hypocotyl). Error bars indicate SD. Columns followed by the same letter are not significantly different (P<0.01). Bars, 100 µm. The length of representative cells is indicated by ‘l’. (This figure is available in colour at JXB online.)

Fig. 3.

AtRALF1-inducible genes. (A, B) Semi-quantitative RT-PCR (A) and qRT-PCR gene expression analyses (B) performed in roots of 10-d-old AtRALF1-overexpressing (35S:AtRALF1), AtRALF1-silenced (irAtRALF1) transgenic lines and wild-type (WT) plants. Error bars indicate SD. Columns followed by the same letter are not significantly different (P<0.01). (C) Semi-quantitative RT-PCR gene expression analyses performed in roots of _HisAtRALF1-treated 10-d-old wild-type plants. Total RNA was extracted from roots of plants after 30min of treatment with different concentrations of the peptide. GAPDH expression was used as a control. AtPRP1 and AtPRP3, proline-rich proteins 1 and 3 (AtPRP1, At1g54970 and AtPRP3, At3g62680); AtHRGP2, hydroxyproline-rich glycoprotein (At5g19800); TCH4, XET gene TOUCH4 (At5g57560); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (At1g13440). All experiments were repeated at least three times (independent biological replicates).

Fig. 4.

AtRALF1-overexpressing (35S:AtRALF1, black columns), AtRALF1-silencing (irAtRALF1, grey columns) transgenic lines and wild-type (WT) plants (white columns) treated with different concentrations of BL. (A) Root length. (B) Hypocotyl length of light-grown plants. (C) Number of emerged lateral roots per cm. Root length, hypocotyl length, and number of emerged lateral roots were measured in 10-d-old seedlings (n>30) grown in the presence of different concentrations of BL. Error bars indicate SD. Statistical analysis was made for each genetic background separately. Columns within each genotype that are followed by the same letter are not significantly different (P<0.01). All experiments were repeated at least three times.

Fig. 5.

AtRALF1 effect on hypocotyl elongation in the presence or absence of BL at high concentrations. (A) Hypocotyl length of light-grown 5-d-old seedlings _HisAtRALF1 treated (1 µM, grey columns) or untreated (control, white columns). (B) Hypocotyl elongation of dark-grown 5-d-old seedlings _HisAtRALF1 treated (1 µM, grey columns) or untreated (control, white columns). Error bars indicate SD. Within each concentration of BL, columns followed by the same letter are not significantly different (P<0.01). All experiments were repeated at least three times.

Fig. 6.

AtRALF1 effect on BL-downregulated genes CPD (At5g05690) and DWF4 (At3g50660). (A, B) Semi-quantitative RT-PCR gene expression analyses performed in roots of 10-d-old AtRALF1-overexpressing (35S:AtRALF1) and AtRALF1-silenced (irAtRALF1) transgenic lines and wild-type plants (A) and quantitative qRT-PCR gene expression analyses (B). Error bars indicate SD. Columns followed by the same letter are not significantly different (P<0.01). (C) Semi-quantitative RT-PCR gene expression analyses performed in roots of _HisAtRALF1-treated 10-d-old wild-type plants. Total RNA was extracted from roots of plants after 30min of treatment with different concentrations of the peptide. GAPDH (At1g13440) expression was used as a control. All experiments were repeated at least three times (independent biological replicates).

Fig. 7.

Gene expression analyses of AtRALF1-inducible genes upon simultaneous treatment with _HisAtRALF1 (R, 1 μM for 30min) and BL (1 μM for 30min). (A) Semi-quantitative RT-PCR performed using total RNA extracted from roots of untreated (C, control plants) or treated (R, BL, or R+BL) 10-d-old wild-type (WT) or BL-insensitive mutant bri1 plants. (B) qRT-PCR performed using total RNA extracted from roots of untreated (C, control plants) or treated (R, BL, or R+BL) 10-d-old wild-type (WT) or BL-insensitive mutant bri1 plants. GAPDH expression was used as a control. AtPRP1 and AtPRP3, proline-rich proteins 1 and 3 (AtPRP1, At1g54970, and AtPRP3, At3g62680); AtHRGP2, hydroxyproline-rich glycoprotein (At5g19800); TCH4, xyloglucan endotransglucosylase TOUCH4 (At5g57560); CPD, constitutive photomorphism and dwarfism (At5g05690). DWF4, DWARF4 (At3g50660); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (At1g13440). DWF4 and AtPRP1 were amplified using 26 and 29 PCR cycles, respectively. Error bars indicate SD. Statistical analysis was made for each genetic background separately. Columns within each genotype that are followed by the same letter are not significantly different (P<0.01). The experiment was performed at least three times (independent biological replicates).