MtABCG20 is an ABA exporter influencing root morphology and seed germination of Medicago truncatula

Abstract

Abscisic acid (ABA) integrates internal and external signals to coordinate plant development, growth and architecture. It plays a central role in stomatal closure, and prevents germination of freshly produced seeds and germination of non-dormant seeds under unfavorable circumstances. Here, we describe a Medicago truncatula ATP-binding cassette (ABC) transporter, MtABCG20, as an ABA exporter present in roots and germinating seeds. In seeds, MtABCG20 was found in the hypocotyl-radicle transition zone of the embryonic axis. Seeds of mtabcg20 plants were more sensitive to ABA upon germination, due to the fact that ABA translocation within mtabcg20 embryos was impaired. Additionally, the mtabcg20 produced fewer lateral roots and formed more nodules compared with wild-type plants in conditions mimicking drought stress. Heterologous expression in Arabidopsis thaliana provided evidence that MtABCG20 is a plasma membrane protein that is likely to form homodimers. Moreover, export of ABA from Nicotiana tabacum BY2 cells expressing MtABCG20 was faster than in the BY2 without MtABCG20. Our results have implications both in legume crop research and determination of the fundamental molecular processes involved in drought response and germination.

Keywords: ABC transporters; abscisic acid; germination; legumes; root organ formation.

Figure 1

Expression analyses of MtABCG20 in Medicago truncatula roots. Quantitative polymerase chain reaction (qPCR) time‐course expression analysis was performed for MtABCG20 in roots treated with (a) 15% polyethylene glycol [PEG; real‐time (RT)‐PCR] or (b) 10 μm abscisic acid (ABA; Droplet Digital PCR). The transcript levels were normalized to the Actin gene. The data represent the mean ± SD of two independent biological experiments and three technical repeats. Significant differences from the control plants determined by Student's t‐test are indicated: **P < 0.01. (c) Promoter activity analyses of MtABCG20 in transgenic M. truncatula roots. Expression of ProMtABCG20:GUS in the root (left panel) and root cross‐section (right panel). (d) Expression of ProMtABCG20:NLS‐GFP. Fluorescence images (left panel) and the merging of fluorescence and brightfield images (right panel).

Figure 2

Phenotypic characterization of mtabcg20 mutants. (a) Schematic diagram indicating Tnt1 insertions in two mtabcg20 mutants, mtabcg20‐1 (NF10694) and mtabcg20‐2 (NF6539). Light gray and dark gray boxes indicate exons and introns of MtABCG20, respectively. (b) Full‐length MtABCG20 mRNA in mutant lines analyzed by reverse transcription (RT)‐polymerase chain reaction (PCR). Actin used as an internal control. (c) Average lateral root (LR) number per plant in wild‐type (WT) and mtabcg20 plants. All plants were grown for 4 weeks on ½ Murashige and Skoog (MS) medium containing 5% polyethylene glycol (PEG). Data represent the mean ± SD of three independent biological experiments on 30 plants (Student's t‐test *P < 0.05). (d) Average nodule number per plant in WT and mtabcg20 plants. Three‐day‐old seedlings, pre‐treated with 10 μm abscisic acid (ABA), were inoculated with Sinorhizobium meliloti and grown on modified Fahraeus (‐N) medium. At 21 days post‐inoculation (dpi), nodule numbers were counted. The data represent the mean ± SD of two independent biological experiments with five technical repeats (eight plants each), per line (Student's t‐test *P < 0.05). (e) Real‐time PCR expression analyses of MtNCED in roots derived from WT‐2 and mtabcg20‐2, untreated or treated with 15% PEG. Transcript levels were normalized to the Actin gene. The data represent the mean ± SD of two independent biological experiments and three technical repeats. Significant differences between the groups were determined by Bonferroni post hoc tests following two‐way anova with the factors of genotype and condition: *P < 0.05, ***P < 0.001. (f) Semi‐quantitative PCR analyses of ABA‐dependent induction of MtABCG20, MtNCED and MtGPAT5 in M. truncatula hairy‐root cultures transformed with empty vector (EV) or overexpressing abi1‐1, 24 h after 10 μm ABA treatment. Abi1‐1 primers were used to confirm abi1‐1 allele expression in M. truncatula transgenic roots. The Actin transcript was used as an internal control.

Figure 3

MtABCG20 plasma membrane localization and homodimer formation. (a) Co‐localization of fused green fluorescent protein (GFP)‐MtABCG20 and mCherry‐labeled plasma membrane marker AtPIP2A in Arabidopsis mesophyll protoplast. (b) Bimolecular fluorescent complementation (BiFC) assay demonstrating interaction of two MtABCG20 half‐size transporters. The fusion proteins Venus‐MtABCG20 and MtABCG20‐CFP were transiently expressed in Arabidopsis mesophyll protoplasts. Scale bar: 10 μm.

Figure 4

Abscisic acid (ABA) transport assays in BY2 cells and cell‐derived vesicles. (a) ABA efflux from BY2 control (EV) and MtABCG20‐overexpressing cell lines, conducted at 22°C and monitored by HPLC/MS. The 100% represents a quantity of cell‐associated ABA, defined as the ratio of the single‐ion chromatogram peak area to the internal standard, at time 0 (T0). Values represent the mean of three independent experiments ± SD. Significant differences between control and overexpressing lines determined by Student's t‐test are indicated: *P < 0.05, **P < 0.01. (b) Transport of ³H‐ABA into membrane vesicles derived from BY2 cells overexpressing MtABCG20 in the absence of ATP as well as in the presence of ATP with or without orthovanadate. Values represent the mean of three independent experiments ± SD. Significant differences between ³H‐ABA uptake in the presence of ATP in comparison to other conditions were determined by an anova test and Tukey's multiple comparison test, and are as follows: **P < 0.01, ***P < 0.005.

Figure 5

Expression analyses of MtABCG20 in Medicago truncatula seeds. (a) Quantitative polymerase chain reaction (qPCR) time‐course expression analysis of MtABCG20 during seed germination. Data represent the mean ± SD of two independent biological experiments and three technical repeats. Transcript levels were normalized to the Actin gene. Images below the graph show the Medicago seeds at different time points during germination. (b) MtABCG20 promoter activity analyses in M. truncatula seeds using the β‐glucuronidase (GUS) reporter system. Seeds were stained for GUS activity and, consequently, particular seed parts (testa, endosperm, end embryo) were separated and visualized by light microscopy. *hypocotyl–radicle transition zone of embryo.

Figure 6

Seed germination phenotype of the mtabcg20 mutant. (a) Germination assay of wild‐type (WT) and mtabcg20 seeds. Stratified seeds were imbibed in the presence of different concentrations of abscisic acid (ABA) for 3 days at 4°C in the dark, and then moved to 23°C and scored 24 h after stratification. Each value represents the average percentage of germination of 30 seeds ± the SD of three replicates. Asterisks indicate significant differences of each mtabcg20 line compared with WT based on Student's t‐test (*P < 0.05). (b) Real‐time polymerase chain reaction (PCR) expression analyses of MtHAI2 and MtEXP1 in embryo axes derived from WT and mtabcg20 dissected embryos, untreated or treated with ABA applied onto the hypocotyl–radicle region. Transcript levels were normalized to the Actin gene. Results are presented as mean ± SD of three technical replicates of one representative biological repeat. Significant differences from the WT plants determined by Student's t‐test are indicated: *P < 0.05, **P < 0.01, ***P < 0.001. (c) Accumulation of ³H‐ABA in the embryo axis of WT and mtabcg20 dissected embryos over 2 h; 1 μl of ³H‐ABA (0.15 mCi mmol⁻¹) was applied to the embryo axis. Data are means ± SD of n = 30. Significant differences from the control (WT) determined by Student's t‐test are indicated: ***P < 0.001.

Figure 7

A proposed role of the MtABCG20 in Medicago roots and seeds.(a) MtABCG20 is an abscisic acid (ABA) exporter from biosynthesizing cells in roots (vascular parenchyma cells) enabling delivery of this hormone to the place where ABA‐dependent responses occur. In this way, MtABCG20 could positively affect lateral root (LR) primordium formation and exert a negative effect on the development of nodule primordia in Medicago. (b) In seeds MtABCG20 is responsible for extrusion of ABA from the hypocotyl–radicle transition zone, thereby facilitating germination.