Haem oxygenase modifies salinity tolerance in Arabidopsis by controlling K⁺ retention via regulation of the plasma membrane H⁺-ATPase and by altering SOS1 transcript levels in roots

Abstract

Reactive oxygen species (ROS) production is a common denominator in a variety of biotic and abiotic stresses, including salinity. In recent years, haem oxygenase (HO; EC 1.14.99.3) has been described as an important component of the antioxidant defence system in both mammalian and plant systems. Moreover, a recent report on Arabidopsis demonstrated that HO overexpression resulted in an enhanced salinity tolerance in this species. However, physiological mechanisms and downstream targets responsible for the observed salinity tolerance in these HO mutants remain elusive. To address this gap, ion transport characteristics (K(+) and H(+) fluxes and membrane potentials) and gene expression profiles in the roots of Arabidopsis thaliana HO-overexpressing (35S:HY1-1/2/3/4) and loss-of-function (hy-100, ho2, ho3, and ho4) mutants were compared during salinity stress. Upon acute salt stress, HO-overexpressing mutants retained more K(+) (less efflux), and exhibited better membrane potential regulation (maintained more negative potential) and higher H(+) efflux activity in root epidermis, compared with loss-of-function mutants. Pharmacological experiments suggested that high activity of the plasma membrane H(+)-ATPase in HO overexpressor mutants provided the proton-motive force required for membrane potential maintenance and, hence, better K(+) retention. The gene expression analysis after 12h and 24h of salt stress revealed high expression levels of H(+)-ATPases (AHA1/2/3) and Na(+)/H(+) antiporter [salt overly sensitive1 (SOS1)] transcripts in the plasma membrane of HO overexpressors. It is concluded that HO modifies salinity tolerance in Arabidopsis by controlling K(+) retention via regulation of the plasma membrane H(+)-ATPase and by altering SOS1 transcript levels in roots.

Fig. 1.

(A) Overview of the haem oxygenase (HO) knockout and overexpression Arabidopsis thaliana lines used in this study. (B) Percentage of radicle emergence in 100mM NaCl agar medium 48h after sowing. (C) Fresh weight of Arabidopsis seedlings grown in 50mM NaCl agar medium. In B and C, each bar represents the mean ±SE of two independent experiments. Bars sharing common letters are not significantly different by LSD test at P ≤ 0.05. (This figure is available in colour at JXB online.)

Fig. 2.

Effect of 100mM NaCl stress on H⁺ fluxes measured at the mature root zone of 4- to 5-day-old Arabidopsis seedlings. (A) Comparison between the wild type and HO overexpressor mutants. (B) Comparison between the wild type and HO knockout mutants. (C) Average H⁺ extrusion during 1h of 100mM NaCl stress. Each point or bar represents the mean ±SE (n=6–9 seedlings). In C, bars sharing common letters are not significantly different by LSD test at P ≤ 0.05. (This figure is available in colour at JXB online.)

Fig. 3.

Effect of 100mM NaCl stress on H⁺ fluxes measured at the elongation root zone of 4- to 5-day-old Arabidopsis seedlings. (A) Transient H⁺ flux comparison between the wild type, and HO overexpressor and suppressor mutants. (B) Average H⁺ extrusion during 1h of 100mM NaCl stress. Each point or bar represents the mean ±SE (n=6–9 seedlings). In B, bars sharing common letters are not significantly different by LSD test at P ≤ 0.05. (This figure is available in colour at JXB online.)

Fig. 4.

Effect of 100mM NaCl treatment on transient (A) H⁺ and (B) K⁺ flux kinetics measured at the elongation zone of the 35s:HY1-4 mutant in the presence and absence of the H⁺-pump inhibitor vanadate. Each point represents the mean±SE (n=6–9 seedlings).

Fig. 5.

Effect of 100mM NaCl stress on transient K⁺ efflux measured at (A) the mature and (B) the elongation root zone of 4- to 5-day-old Arabidopsis seedlings. (C) Average K⁺ extrusion during 1h of 100mM NaCl stress. Each point or bar represents the mean±SE (n=6–9 seedlings). In C, bars sharing common letters are not significantly different by LSD test at P ≤ 0.05. (This figure is available in colour at JXB online.)

Fig. 6.

Effect of 100mM NaCl treatment on transient membrane potential kinetics measured at the elongation root zone of 4- to 5-day-old Arabidopsis seedlings. Each point or bar represents the mean ±SE (n=6–9 seedlings).

Fig. 7.

Effect of 100mM NaCl treatment on relative SOS1 and AHA1/2/3 expression measured in roots of 4- to 5-day-old Arabidopsis seedlings. Each bar represents the mean ±SE (n=6–9 seedlings for each treatment and time combination obtained in three independent trials).

Fig. 8.

Schematic diagram comparing Arabidopsis thaliana haem oxygenase (HO) loss- and gain-of-function mutants during salinity stress. SOS1, salt overly sensitive1 Na⁺/H⁺ exchanger; KOR, potassium outward rectifying channels; NSCC, non-selective cation channels; AHA, P-type ATPases. AHAs in shades of grey are additional copies after transcriptional regulation. Ticks denote up-regulation and crossess denote down-regulation of a respective transporter. (This figure is available in colour at JXB online.)