ABA-mediated inhibition of germination is related to the inhibition of genes encoding cell-wall biosynthetic and architecture: modifying enzymes and structural proteins in Medicago truncatula embryo axis

Abstract

Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 microM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of alpha-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.

Keywords: ABA; Medicago truncatula; XET; cell-wall expansion; germination; radicle emergence.

Figure 1.

Effect of Permanent and Transient Abscisic Acid Treatment on Medicago truncatula Seed Germination and Radicle Growth. Germination rates (A) and radicle length (B) are the mean of 150 measurements taken on 150 seeds germinated on water, 100 μM ABA or 100 μM ABA during 15 h before transfer of the seeds on water.

Figure 2.

Microscopic Observation of Longitudinal Sections of Medicago truncatula Embryos at Two Developmental Stages. Imbibed seed (5 h) before germination completion (A) and after germination completion and radicle emergence (21 h) (B). ×4 enlargement shows details of cells clustered in spindles. Boxes correspond to the apical region (1) and the upper region (2) of the radicle. Int, internal cell layers; Ext, external cell layers; CP, cortical parenchyma; V, vascular tissues; RC, root cap.

Figure 3.

Changes in Cortical Parenchyma Cell Length (A) and Anatomy (B) in the Apical and Upper Region of Medicago truncatula Radicle at Various Developmental Stages, Before and After Germination Completion. Pictures are presented on highly saturated inverted colors. ×40. CP, cortical parenchyma; RC, root cap.

Figure 4.

Effect of 10 μM Oryzalin on Medicago truncatula Seed Germination and Seedling Post-Germination Growth. O, oryzalin condition; W, water condition.

Figure 5.

Microscopic Observation of a Longitudinal Section of Medicago truncatula Embryo Axis Cells after Germination Completion and Radicle Emergence after 25 h of Oryzalyn (10 μm) Treatment. Hypocotyl (A), upper region (B), sub-apical region (C), and apical region (D) of the radicle. Pictures are presented on highly saturated inverted colors. ×40. RC, root cap; CP, cortical parenchyma; E, epidermis.

Figure 6.

Expression of Genes Involved in Cell-wall Biosynthesis and Architecture Modification and Cell Cycle in Medicago truncatula Embryo Axis under Various Conditions of Germination: Water, Permanent, and Transient (100 μM) ABA and 10 μM Oryzalin Treatment. The results are the mean ± SE of at least two biological replicates with duplicated PCR reaction for determining Ct values. Expression levels are presented in arbitrary units.

Figure 6.

Expression of Genes Involved in Cell-wall Biosynthesis and Architecture Modification and Cell Cycle in Medicago truncatula Embryo Axis under Various Conditions of Germination: Water, Permanent, and Transient (100 μM) ABA and 10 μM Oryzalin Treatment. The results are the mean ± SE of at least two biological replicates with duplicated PCR reaction for determining Ct values. Expression levels are presented in arbitrary units.