Aminocyclopropane-1-carboxylic acid is a key regulator of guard mother cell terminal division in Arabidopsis thaliana

Abstract

Stomata have a critical function in the exchange of gases and water vapor between plants and their environment. Stomatal development is under the rigorous control of many regulators. The last step of development is the terminal division of guard mother cells (GMC) into two guard cells (GC). It is still unclear how the symmetric division of GMCs is regulated. Here, we show that the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) is required for the symmetric division of GMCs into GCs in Arabidopsis. Exogenous application of the ACC biosynthesis inhibitor aminoethoxyvinylglycine (AVG) induced the formation of single guard cells (SGCs). Correspondingly, an acs octuple-mutant with extremely low endogenous ACC also developed SGCs, and exogenous ACC dramatically decreased the number of SGCs in this mutant whereas exogenous ethephon (which is gradually converted into ethylene) had no effect. Furthermore, neither blocking of endogenous ethylene synthesis nor disruption of ethylene signaling transduction could induce the production of SGCs. Further investigation indicated that ACC promoted the division of GMCs in fama-1 and flp-1myb88 mutants whereas AVG inhibited it. Moreover, ACC positively regulated the expression of CDKB1;1 and CYCA2;3 in the fama-1 and flp-1myb88 mutants. The SGC number was not affected by ACC or AVG in cdkb1;11;2 and cyca2;234 mutants. Taken together, the results demonstrate that ACC itself, but not ethylene, positively modulates the symmetric division of GMCs in a manner that is dependent on CDKB1s and CYCA2s.

Fig. 1.

Treatment with aminoethoxyvinylglycine (AVG) results in the formation of single guard cells (SGCs). (A) Abaxial epidermis of cotyledons in the wild-type (WT) with or without AVG treatment. Arrows indicate SGCs. Scale bars are 20 μm. (B) E1728 expression in the abaxial epidermis of cotyledons in the wild-type before and after AVG treatment. Arrow indicates SGCs. Scale bars are 100 μm. (C) Abaxial epidermis of a cotyledon in the wild-type with AVG treatment. The confocal z-projection was visualized using propidium iodide staining with a confocal laser-scanning microscope. The yellow arrow indicates a SGC without a central cell wall. White arrows indicate normal stomata with two GCs and a thickened central cell wall. The scale bar is 50 μm. (D) GCs and SGCs with DAPI fluorescence. Scale bars are 5 μm. (E) Quantitative analysis of DAPI fluorescence intensity in GCs and SGCs. The data are means (±SD) (n=10) and the significant difference between SGCs and GCs was determined using Student’s t-test: *P<0.01. (F) Frequency of cotyledons with different numbers of SGCs on the abaxial epidermis of AVG-treated wild-type plants. A total of 50 cotyledons from 25 seedlings were examined. (G) Mean number of SGCs per cotyledon in the wild-type with or without (Mock) AVG treatment. The significant difference between the means was determined using Student’s t-test: *P<0.01. A total of 50 cotyledons from 25 seedlings were examined.

Fig. 2.

Defect of guard mother cell (GMC) division in the Arabidopsis asc octuple-mutant. (A) Abaxial epidermis of cotyledons of the wild-type (WT) and acs octuple-mutant. The arrow indicates a single guard cell (SGC). Scale bars are 20 μm (B) E1728 expression in the abaxial epidermis of cotyledons of the wild-type and acs octuple-mutant. The arrow indicates a SGC. Scale bars are 100 μm. (C) Abaxial epidermis of a cotyledon of the acs octuple-mutant. The confocal z-projection image was visualized using propidium iodide staining with a confocal laser-scanning microscope. The yellow arrow indicates a SGC without a central cell wall. White arrows indicate normal stomata with two GCs and a thickened central cell wall. Scale bar is 50 μm. (D) GCs and SGCs imaged with DAPI fluorescence. Sacle bars are 5 μm. (E) Quantitative analysis of DAPI fluorescence intensity in GCs and SGCs. The data are means (±SD) (n=10) and the significant difference between SGCs and GCs was determined using Student’s t-test: *P<0.01. (F) Number of SGCs per cotyledon in the acs octuple-mutant in control (Mock) plants and plants treated with aminocyclopropane-1-carboxylic acid (ACC) and aminoethoxyvinylglycine (AVG). The data are means (±SD) and significant differences compared with the Mock were determined using Student’s t-test: *P<0.01. A total of 50 cotyledons from 25 seedlings were analysed.

Fig. 3.

Blocking ethylene synthesis and signaling has no effect on guard mother cell division in Arabidopsis wild-type (WT) and acs octuple-mutant plants. (A) Abaxial epidermis of the wild-type after treatment with α-aminoisobutyric acid (AIB), Co²⁺, or 1-methycyclopropene (1-MCP) compared with untreated controls (Mock). Scale bars are 20 μm. (B) Numbers of single guard cells (SGCs) of abaxial epidermis of the acs octuple-mutant after treatment with ethephon. Arrow indicates SGCs. Scale bars are 20 μm. Data are are means (±SD). (C) Abaxial epidermis of the ethylene signaling-associated mutants etr1-7, ctr1, ein2, and ein3eil1. Scale bars are 20 μm.

Fig. 4.

Elevation of aminocyclopropane-1-carboxylic acid (ACC) and ethylene fails to induce extra division of guard mother cells in wild-type (WT) Arabidopsis seedlings. (A) Abaxial epidermis of cotyledons. Seedlings were grown on media with ACC or ethephon under a 16/8 h photoperiod or under constant darkness. Scale bars are 20 μm. (B) Abaxial epidermis of cotyledons of the wild-type and the eto1-1 mutant grown under constant darkness. Scale bars are 20 μm.

Fig. 5.

Aminocyclopropane-1-carboxylic acid (ACC) promotes symmetric division of guard mother cells in Arabidopsis fama-1 and flp-1myb88 mutants. (A) Abaxial epidermis of cotyledons of the mutants treated with ACC or aminoethoxyvinylglycine (AVG) compared with the control (Mock). Scale bars are 20 μm. (B) Frequency of cell clusters containing different numbers of cells on the abaxial epidermis of fama-1 and flp-1myb88 mutants treated with ACC or AVG.

Fig. 6.

Aminocyclopropane-1-carboxylic acid (ACC) positively regulates expression of CDKB1;1 and CYCA2;3 in Arabidopsis. (A) Relative mRNA levels of CDKB1;1 and CYCA2;3 (normalized to KAT1) in the wild-type (WT) and fama-1 and flp-1myb88 mutants treated with ACC or aminoethoxyvinylglycine (AVG) compared with the control (Mock). Data are means (±SD) of three biological replicates. Significant differences compared with the corresponding Mock value were determined using Student’s t-test: *P<0.01. Three independent experiments were performed. (B) Expression of pCDKB1;1::GFP and pCYCA2;3::GFP. Representative images are shown. Arrows indicate newly formed GCs. The graphs show quantitative analyses of GFP-positive cells. Data are means (±SD) of three biological replicates. Significant differences compared with the Mock were determined using Student’s t-test: *P<0.01. A total of 50 cotyledons from 25 seedlings were analysed. Three independent experiments were performed.

Fig. 7.

The cdkb1;11;2 and cyca2;234 mutants are insensitive to treatment with aminocyclopropane-1-carboxylic acid (ACC) and aminoethoxyvinylglycine (AVG). (A) Abaxial epidermis of cotyledons of the cdkb1;11;2 and cyca2;234 mutants treated with ACC or AVG compared with the control (Mock). The arrows indicate single guard cells (SGCs). Scale bars are 20 μm. (B) Proportion of SGCs in the cdkb1;11;2 and cyca2;234 mutants and the wild-type (WT) treated with either ACC or AVG. Data are means (±SD) (n=10).

Fig. 8.

Model of the function of aminocyclopropane-1-carboxylic acid (ACC) in the symmetric division of guard mother cells (GMCs). (A) Under normal conditions, ACC and FAMA/FLP/MYB88 maintain the appropriate expression of CDKB1;1 and CYCA2;3 in GMCs and ensure their symmetric division into GCs. (B) When ACC synthesis is blocked, the expression of CDKB1;1 and CYCA2;3 is inhibited, which disrupts the symmetric division of GMCs and then leads to the formation of single guard cells (SGCs).