MADS-box transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals

Abstract

Plant root system morphology is dramatically influenced by various environmental cues. The adaptation of root system architecture to environmental constraints, which mostly depends on the formation and growth of lateral roots, is an important agronomic trait. Lateral root development is regulated by the external signals coordinating closely with intrinsic signaling pathways. MADS-box transcription factors are known key regulators of the transition to flowering and flower development. However, their functions in root development are still poorly understood. Here we report that AGL21, an AGL17-clade MADS-box gene, plays a crucial role in lateral root development. AGL21 was highly expressed in root, particularly in the root central cylinder and lateral root primordia. AGL21 overexpression plants produced more and longer lateral roots while agl21 mutants showed impaired lateral root development, especially under nitrogen-deficient conditions. AGL21 was induced by many plant hormones and environmental stresses, suggesting a function of this gene in root system plasticity in response to various signals. Furthermore, AGL21 was found positively regulating auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. We propose that AGL21 may be involved in various environmental and physiological signals-mediated lateral root development and growth.

Keywords: AGL21; MADS; auxin; lateral root; nitrate; root system architecture; sulfate..

Figure 1

AGL21 Is Involved in LR Development. The seeds were germinated for 5 d on MS medium, and the seedlings were then transferred to MS medium for vertical growth. (A) AGL21 gene structure with the sites of T-DNA insertion. Squares correspond to exons while lines represent introns. (B) AGL21 transcript levels in the transgenic lines and mutants by RT–PCR analysis. TUBULIN (TUB) was used as the internal control. (C) Root systems of 12-day-old 35S::AGL21, agl21 mutants and wild-type (WT) (Col-0) seedlings (bar = 1cm). (D) Density of visible LRs of 12-day-old plants. Density of visible LRs is defined as visible LR number per cm PR. Values are mean ± standard deviation (SD) of three independent experiments each containing 15–20 plants per genotype. Asterisks denote Student’s t-test significance compared with WT plants: * P < 0.05; ** P < 0.01; *** P < 0.001. (E) Average LR length of 12-day-old plants. Average LR length is defined as the ratio of total LR length over LR number. Values are the mean ± SD of three independent experiments each containing 15–20 plants per genotype. Asterisks denote Student’s t-test significance compared with WT plants: * P < 0.05. (F, G) LR and PR growth curves of WT, agl21 mutants, and 35S::AGL21 plants. (H) Numbers of LRP of 8-day-old seedlings at given stages. Stages of primordia were based on the classification by Malamy and Benfey (1997). Values are mean ± SD of three independent experiments each containing 15 plants per genotype and asterisks denote Student’s t-test significance compared with WT plants: * P < 0.05; ** P < 0.01; *** P < 0.001. NE, non-emerged LR; E, emerged LR; T, NE + E

Figure 2

AGL21 Expression Pattern. (A) Analysis of the AGL21 expression pattern in different organs by qRT–PCR. UBQ5 was used as an internal control. Values are mean ± SD of three replica experiments. (B–I) The expression pattern of AGL21, as revealed by promoter–GUS fusion analyses in pAGL21::GUS transgenic seedlings. GUS activity was observed in embryo (B), seedling of 2-day-old (C), seedling of 3-day-old (D), seedling of 4-day-old (E), seedling of 14-day-old (F), seedling of 35-day-old (G), silique (H), and flower (I). (J–S) The pAGL21::GUS expressed in LRPs at stages I to VIII (J–Q), emerged LR (R), and PR tip (S). Eight-day-old pAGL21::GUS transgenic lines were used for GUS reaction for 12h.

Figure 3

Subcellular Localization of AGL21 Protein. (A, B) Fluorescence in the root cells of transgenic plants expressing AGL21–GFP under the control of the CaMV 35S promoter (bar = 50 μm). (C) Fluorescence in the root cells of transgenic plants expressing AGL21–GFP under the control of the 3.6-kb AGL21 promoter (bar = 50 μm).

Figure 4

AGL21 Expression Is Regulated by Hormones. (A–C) qRT–PCR analyses of AGL21 expression in wild-type seedlings during the time course after IAA (A), MeJA (B), or ABA (C) treatment. Eight-day-old Col-0 seedlings were incubated in MS liquid cultures with 10 μM IAA, 50 μM MeJA, and 20 μM ABA, respectively, and whole seedlings were harvested at indicated time points for RNA extraction and qRT–PCR analyses. The transcript levels of AGL21 were normalized to the UBQ5 expression. Values are mean ± SD and asterisks denote Student’s t-test significance compared with untreated plants: * P < 0.05; ** P < 0.01; *** P < 0.001. (D, E) IAA, MeJA, and ABA-induced pAGL21::GUS expression in the primary root (D), LRP, and LR (E). Seven-day-old seedlings of pAGL21::GUS transgenic line grown on MS agar medium were transferred either to hormone-free MS agar medium or to MS agar medium supplemented with 10 μM IAA, 10 μM MeJA, or 10 μM ABA for 1 d, respectively. The seedlings were harvested for GUS staining for 8h.

Figure 5

Response of AGL21 to Multiple Environmental Stresses. (A) Response of AGL21 to N starvation. Seven-day-old Col-0 seedlings were transferred to N-free nutrient solution and harvested at indicated time points for RNA extraction and qRT–PCR analyses. (B) Response of AGL21 to S starvation. Seven-day-old Col-0 seedlings were transferred to S-free agar medium for vertical growth. Complete nutrient medium was used as control. Roots were harvested at indicated time points for RNA extraction and qRT–PCR analyses. (C) qRT–PCR analyses of AGL21 expression in 8-day-old Col-0 seedlings after 2.5h of drought or 120mM NaCl treatment. The transcript levels of AGL21 were normalized to the UBQ5 expression. Values are mean ± SD of three replica experiments and asterisks denote Student’s t-test significance compared with the 0-h control plants: * P < 0.05; ** P < 0.01; *** P < 0.001. (D–F) Effects of N deprivation and S deprivation on pAGL21::GUS expression in the roots. Expression of AGL21 is induced by N deprivation and S deprivation in the PR tips (D), differential zone (E) after 1–4 d of N or S starvation, and LRPs after 3 d of N or S starvation (F). Five-day-old pAGL21::GUS transgenic seedlings grown on MS medium were transferred to N- or S-free medium for 1–4 d and seedlings were harvested at the indicated time points for GUS staining for 8h.

Figure 6

AGL21 Is Important for Sustaining LR Development under Low-N Conditions. Five-day-old seedlings grown on MS medium were transferred to N-free medium and grown vertically. During the vertical growth stage, the root morphological parameters were measured. (A) Phenotype of 11-day-old seedlings of AGL21-overexpressing, Col-0, and AGL21 knockout (bar = 1cm). (B, C) Numbers of visible lateral roots of AGL21-overexpressing, Col-0, and AGL21 knockout plants on MS medium (B) and N-free medium (C) from the 7th day to the 10th day. Values are mean ± SD of three independent experiments each containing 15–20 plants per genotype. (D) Average PR length of the 11-day-old plants. Values are mean ± SD of three independent experiments each containing 15–20 plants per genotype. (E–G) Length of visible LRs per cm PR length (E), LR density (F), and average LR length (G). Average LR length defined as the ratio of total LR length over LR number. Values are mean ± SD of three independent experiments each containing 15–20 plants per genotype and asterisks denote Student’s t-test significance compared with the wild-type plants: * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 7

LR Phenotype Is Rescued by Exogenous IAA. Five-day-old plants grown on MS vertical agar plates were transferred to MS agar plates containing different concentrations of IAA to grow vertically for 6 d. Values are mean ± SD of three independent experiments each containing 15 plants. (A) Time course of LR development on MS medium. (B) Time course of LR development on MS medium supplemented with 10nM IAA. (C) Time course of LR development on MS medium supplemented with 50nM IAA.

Figure 8

AGL21 Regulates Auxin Accumulation in the LRPs and LRs. (A) Quantification of free IAA content in the root of 9-day-old plants. Values are mean ± SD of three replica experiments and asterisks denote Student’s t-test significance compared with the wild-type plants: * P < 0.05; ** P < 0.01. (B–F) Expression of DR5:GUS (N = 20 plants) in three different stages of LRP (B–D), LR, and PR tips (E, F) of Col-0 plants. Nine-day-old plants grown on MS medium were used for GUS staining for 6h. (G–K) Expression of DR5:GUS in three different stages of LRP (G–I), LR, and PR tips (J, K) of agl21 knockout plants. (L–P) Expression of DR5:GUS in three different stages of LRP (L–N), LR, and PR tips (O, P) of AGL21-overexpressing plants.

Figure 9

AGL21 Increases Auxin Accumulation in the Root through Local Biosynthesis. (A) Effects of auxin transport inhibitors NPA on LR initiation in wild-type, AGL21-overexpressing and mutant plants. Five-day-old seedlings were transferred to medium supplemented with DMSO alone or the auxin transport inhibitor NPA (0.5 μM) dissolved in DMSO. After 7 d of growth, the LRs produced in the new growth were counted on 30 seedlings. Values are mean ± SD of three replica experiments and asterisks denote Student’s t-test significance compared with the wild-type plants: * P < 0.05; ** P < 0.01. (B, C) Relative expression levels of auxin transport genes (B) and auxin biosynthesis genes (C) in the roots of 9-day-old plants. The transcript levels of auxin transport or biosynthesis genes were normalized to the UBQ5 expression. The expression levels of each gene in the wild-type were set as 1.0. Values are mean ± SD of three replica experiments and asterisks denote Student’s t-test significance compared with the wild-type plants: * P < 0.05; ** P < 0.01.

Figure 10

AGL21 Affects pCYCB1;1::GUS Expression in the LRPs and LRs. Eight-day-old seedlings were harvested for GUS staining. The seedlings were immersed in GUS staining buffer and applied vacuum for 2min, and then incubated at 37°C overnight. (A–C) pCYCB1;1::GUS expression in LRPs (A, B) and LR (C) of Col-0 background seedlings. (D–F) pCYCB1;1::GUS expression in LRPs (D, E) and LR (F) of AGL21-overexpressing background seedlings. (G–I) pCYCB1;1::GUS expression in LRPs (G, H) and LR (I) of agl21 knockout background seedlings.