Abscisic acid regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin

Abstract

Understanding the mechanisms regulating root development under drought conditions is an important question for plant biology and world agriculture. We examine the effect of osmotic stress on abscisic acid (ABA), cytokinin and ethylene responses and how they mediate auxin transport, distribution and root growth through effects on PIN proteins. We integrate experimental data to construct hormonal crosstalk networks to formulate a systems view of root growth regulation by multiple hormones. Experimental analysis shows: that ABA-dependent and ABA-independent stress responses increase under osmotic stress, but cytokinin responses are only slightly reduced; inhibition of root growth under osmotic stress does not require ethylene signalling, but auxin can rescue root growth and meristem size; osmotic stress modulates auxin transporter levels and localization, reducing root auxin concentrations; PIN1 levels are reduced under stress in an ABA-dependent manner, overriding ethylene effects; and the interplay among ABA, ethylene, cytokinin and auxin is tissue-specific, as evidenced by differential responses of PIN1 and PIN2 to osmotic stress. Combining experimental analysis with network construction reveals that ABA regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin.

Keywords: Arabidopsis thaliana; PIN proteins; abscisic acid (ABA); auxin; ethylene; hormonal crosstalk, osmotic stress; root development.

Figure 1

Experimental setup shows that osmotic stress leads to reduced Arabidopsis root growth, smaller meristem with fewer cells, and that abscisic acid (ABA) modulates root growth under stress, via increased DELLA. (a) Medium osmolarity of polyethylene glycol‐infused agar, measured with a vapour pressure osmometer 24 h after overlay solution is removed. n = 10. (b) Primary root meristems stained with propidium iodide after 24 h osmotic stress treatment. Arrowheads indicate quiescent centre and approximate end of the meristematic zone. (c) Meristematic cell count (ANOVA P = 0.002) and meristem size (ANOVA P = 0.04) after 24 h osmotic stress treatment. (d) The effect of ABA and the ABA biosynthesis inhibitor fluridon on root growth under osmotic stress (treatment period, 5–7 d after germination (DAG)). Log_e‐transformed two‐factor ANOVA: P (stress) < 0.0001, P (hormone) < 0.0001, P (interaction) = 0.0049. Blue diamonds, no hormone; black triangles, 0.1 μM ABA; yellow circles, 1 μM ABA; red squares, 0.1 μM fluridon. Asterisk indicates a significant effect. (e) proRGA::GFP:RGA under osmotic stress. (f) GFP:RGA fluorescence under osmotic stress. Measured in ImageJ, ANOVA, P = 0.015. U, unstressed (−0.14 MPa); M, moderate stress (−0.37 MPa); S, severe stress (−1.2 MPa). For confocal images, scale bars represent 50 μm. Error bars indicate ± SEM. Lowercase letters indicate significance with a Tukey pairwise comparison.

Figure 2

Arabidopsis abscisic acid (ABA)‐responsive genes (e.g. RD29B) and ABA‐independent stress genes (e.g. DREB2B) are up‐regulated by osmotic stress; cytokinin response genes (ARR5,TCS) may go down slightly. (a) proARR5::GFP (top panels) and pTCS::GFP (bottom panels) after 24 h osmotic stress treatment. (b) RD29B expression under osmotic stress. Log_e‐transformed two‐factor ANOVA: P (stress) < 0.0001, P (time) = 0.42, P (interaction) = 0.15. Red, 6 h treatment; blue, 24 h treatment. (c) DREB2B expression under osmotic stress. Two‐factor ANOVA: P (stress) = 0.0014, P (time) = 0.0014, P (interaction) = 0.3. Red, 6 h treatment; blue, 24 h treatment. (d) pARR5::GFP fluorescence after 24 h osmotic stress treatment. Measured in ImageJ, ANOVA P = 0.0015. (e) ARR5 transcript abundance. (f) pTCS::GFP fluorescence after 24 h osmotic stress treatment, measured in ImageJ. ANOVA, P = 0.44. U, unstressed (−0.14 MPa); M, moderate stress (−0.37 MPa); S, severe stress (−1.2 MPa). For confocal images, scale bars represent 50 μm. Error bars indicate ± SEM. Lowercase letters indicate significance with a Tukey pairwise comparison.

Figure 3

Auxin and ethylene regulation of Arabidopsis root length under osmotic stress. (a) Root growth of Col‐0 (blue diamonds) and the ethylene insensitive mutant ein2 (red squares) under osmotic stress (treatment: 5–8 d after germination (DAG)) Log_e‐transformed two‐factor ANOVA: P (stress) < 0.0001, P (mutant) = 0.71, P (interaction) = 0.063. (b) The effect of 1‐aminocyclopropane‐1‐carboxylic acid (ACC, red squares) and silver thiosulphate (STS, black circles) on root growth under osmotic stress, in Col‐0 (treatment: 5–11 DAG). Blue diamonds, no hormone treatment (NT). Two‐factor ANOVA: P (stress) < 0.0001, P (hormone) < 0.0001, P (interaction) = 0.12. (c) The effect of indole‐3‐acetic acid (IAA) on root growth under osmotic stress. Blue diamonds, no hormone treatment; red squares, 0.1 nM IAA; black circles, 1 nM IAA. Treatment: 5–11 DAG. Log_e‐transformed two‐factor ANOVA: P (hormone) = 0.43, P (stress) < 0.0001, P (interaction) = 0.036. Asterisk indicates a significant effect. (d) The effect of osmotic stress on root growth on wild‐type (Col‐0, blue triangles), auxin transport mutants (eir1‐1/pin2, red squares; aux1‐7, black circles) and an auxin‐resistant mutant (axr3‐1, yellow triangles). Log_e‐transformed two‐factor ANOVA: P (stress) < 0.0001, P (mutant) < 0.0001, P (interaction) < 0.0001. Treatment: 5–8 DAG. Error bars indicate ± SEM. (e) Root meristems under combined IAA and osmotic stress treatments (5–11 DAG). Arrowheads indicate the position of the quiescent centre and the end of the meristematic zone. U, unstressed (−0.14 MPa); M, moderate stress (−0.37 MPa); S, severe stress (−1.2 MPa). Bars, 50 μm. Lowercase letters (a‐d) indicate statistical significance.

Figure 4

Ethylene response to osmotic stress in Arabidopsis. Osmotic stress causes an increased ethylene response, seen as increased expression of ethylene‐responsive genes (e.g ERF1) and suppression of genes down‐regulated by ethylene, such as PLS. (a) Relative transcript abundance of ERF1 after 24 h osmotic stress treatment. ANOVA, P = 0.09. (b) Relative fluorescence of proPLS::PLS:GFP after 24 h osmotic stress treatment. ANOVA, P = 0.23. (c) proPLS::PLS:GFP after 24 h osmotic stress treatment. Green, green fluorescent protein; magenta, propidium iodide. Error bars ± SEM. Scale bars, 50 μm. U, unstressed (−0.14 MPa); M, moderate stress (−0.37 MPa); S, severe stress (−1.2 MPa). Letters indicate significance with a Tukey pairwise comparison.

Figure 5

Response of auxin transport and responses to osmotic stress in Arabidopsis. Osmotic stress modulates auxin transporter levels, reducing root auxin concentrations. (a) pDR5rev::3xVENUS‐N7, 35S::DII:VENUS‐N7, proPIN4::PIN4:GFP, proPIN1::PIN1:GFP, proPIN2::PIN2:GFP and proAUX1::AUX1:YFP after 24 h osmotic stress treatment. (b) DII:VENUS fluorescence under osmotic stress. ANOVA, P = 0.003. (c) Auxin transporter relative expression under osmotic stress. ANOVA: PIN1, P = 0.05; PIN4, P = 0.05; PIN2, P = 0.33; AUX1, P = 0.05. (d) proPIN2::PIN2:GFP fluorescence under osmotic stress. ANOVA, P = 0.003. Lowercase letters indicate significance with a Tukey's pairwise comparison. Green, green fluorescent protein/yellow fluorescent protein; magenta, propidium iodide. Scale bars, 50 μm. Error bars indicate ± SEM. U, unstressed (−0.14 MPa); M, moderate stress (−0.37 MPa); S, severe stress (−1.2 MPa).

Figure 6

Relationship between osmotic stress, ABA and auxin/ethylene. ABA application reduces PIN1 expression further to osmotic stress, and overrides the effect of ethylene in increasing PIN1 levels, indicating that ABA suppresses the ethylene response in the Arabidopsis root. (a) proPIN1::PIN1:GFP under osmotic stress with either the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid (ACC) or the perception inhibitor silver thiosulphate (STS). Green, green fluorescent protein (GFP). (b) proPIN1::PIN1:GFP under osmotic stress with either ABA or the biosynthesis inhibitor fluridon. Green, GFP. (c) proPIN1::PIN1:GFP under combined ACC and ABA treatment. Green is GFP fluorescence, magenta is propidium iodide fluorescence. (d) proPIN1::PIN1:GFP fluorescence under osmotic stress treatment with no hormone (blue bars), 1 μM ACC (green bars) or 10 μM STS (red bars). (e) proPIN1::PIN1:GFP fluorescence under osmotic stress treatment with either no hormone (blue bars), 1 μM ABA (green bars) or 1 μM fluridon (red bars). (f) proPIN1::PIN1:GFP fluorescence under combined ABA and ACC treatment. Error bars indicate ± SEM. U, unstressed (−0.14 MPa); M, moderate stress (−0.37 MPa); S, severe stress (−1.2 MPa). Lowercase letters indicate significance with a Tukey pairwise comparison; ns, no statistical difference; asterisk, a significant difference.

Figure 7

A hormonal crosstalk network for the regulation of root growth under osmotic stress conditions, in a vascular cell expressing PIN1, revealing that abscisic acid (ABA) regulates Arabidopsis root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin. Abbreviations: Ra, inactive auxin receptor; Ra*, active auxin receptor; DR5m, DR5 regulated YFP mRNA transcript; DR5p, DR5 regulated yellow fluorescent protein; DIIp, DII‐VENUS protein; PIN1m, PIN1 mRNA transcript; PIN1p, PIN1 auxin efflux transporter protein; AUX1m, AUX1 mRNA transcript; AUX1p, AUX1 auxin influx transporter protein; PLSm, POLARIS mRNA transcript; PLSp, POLARIS peptide; ET, ethylene; Re, inactive ethylene receptor; Re*, active ethylene receptor; CTR1, inactive CTR1 kinase; CTR1*, active CTR1 kinase; X, the unknown factor that regulates auxin transport from the aerial tissues; ERF1m, ERF1 mRNA transcript ; Raba, inactive ABA receptor; Raba*, active ABA receptor; RD29Bm, RD29B mRNA transcript CK, active cytokinin; Rck, inactive cytokinin receptor; Rck*, active cytokinin receptor. ARR5m, ARR5 mRNA transcript; ARR5p, ARR5 protein; osmotic stress, the osmotic stress imposed by the growth medium.

Figure 8

A simplified representation of the hormonal crosstalk network for the regulation of root growth under osmotic stress conditions, in a vascular cell expressing PIN1, demonstrating that the responses of auxin transporters, hormones and signalling components to osmotic stress are nonlinear and complex. Abbreviations: DR5p, DR5 regulated yellow fluorescent protein; DIIp, DII‐VENUS protein; PLSp, POLARIS peptide; PIN1, PIN1 auxin efflux transporter protein; AUX1, AUX1 auxin influx transporter protein; ET, ethylene; X, the unknown factor that regulates auxin transport from the aerial tissues; EIN2, EIN2 ethylene signalling protein; ERF1m, ERF1 mRNA transcript; ABA, abscisic acid; RD29Bm, RD29B mRNA transcript; CK, active cytokinin; ARR5m, ARR5 mRNA transcript; TCS, cytokinin response reporter; osmotic stress, the osmotic stress imposed by the growth medium. Red boxes group related activities.