Functional analysis of the role of hydrogen sulfide in the regulation of dark-induced leaf senescence in Arabidopsis

Abstract

There is growing evidence that hydrogen sulfide (H₂S) is involved in many physiological processes in plants, but the role of H₂S in dark-induced leaf senescence remains unknown. In this work, we found that H₂S not only inhibited chlorophyll degradation but also caused the accumulation of photoreactive pheide a in detached leaves under extended darkness. Despite this, transcript levels of senescence-associated genes (SAGs) were less affected in H₂S-treated detached leaves compared with those in H₂S-untreated detached leaves. Furthermore, cell death/rapid bleaching occurred in both H₂S-treated detached and attached leaves after transfer from extended darkness to light. Unlike the lack of effect of H₂S on SAG transcripts in darkened detached leaves, exogenous H₂S induced higher SAG transcript levels in attached leaves than untreated attached leaves. Genetic evidence further underlined the positive correlation between SAG expression in attached leaves and H₂S. In addition, effects of H₂S on SAG expression in attached leaves were compromised in the S-nitrosoglutathione reductase-deficient mutant, gsnor1. Taken together, our results suggest that H₂S suppresses chlorophyll degradation of detached leaves by regulating a dark-dependent reaction, and that this gas positively modulates SAG expression in attached leaves under prolonged darkness in a GSNOR1-dependent manner.

Figure 1

Effect of H₂S exposure on chlorophyll breakdown and SAG expression in detached leaves during extended darkness for up to 4 d. (a,b), effects of H₂S gas released from 0 to 2 mM H₂S donor NaHS solution (see Materials and Methods) on leaf yellowing and chlorophyll content, respectively, at 4 d of darkness. Effect of another H₂S donor GYY4137 and H₂S scavenger HT (c) on chlorophyll degradation under extended darkness for 4 d. For GYY4137 and HT treatments, 3-week-old detached leaves were floated in petri dishes containing 3 mL solution of 0.1 mM GYY4137 alone, 0.1 mM HT alone or 0.1 mM GYY4137 plus 0.1 mM HT combined treatment. Transcript levels of SAG12 (d), SAG20 (e) and SEN4 (f) in detached leaves of wild type subjected to H₂S or H₂S-free treatment for up to 4 d of complete darkness. + and − indicate detached leaves of Col-0 fumigated with or without 0.5 mM NaHS respectively. Data are means ± SE of at least three independent samples from different plants. Letters indicates significant difference from the wild type at P < 0.05, using the Student’s t test.

Figure 2

Effects of H₂S exposure on accumulation of green intermediates of chlorophyll breakdown and cell death in detached leaves. Amounts of chlorophyll a (a) and pheophytin a (b) accumulation in detached leaves of wild type subjected to H₂S or H₂S-free treatment for up to 4 d of complete darkness. (c), Accumulation of pheide a in response to dark incubation of detached leaves of control and H₂S-treated wild type for up to 4 d. + and − indicate detached leaves of Col-0 fumigated with or without 0.5 mM NaHS, respectively. (d), Determination of ion leakage as a measure for cell death in detached leaves treated with H₂S (white bars) or without H₂S (black bars). Before re-exposure to the light for up to 12 h, detached leaves were incubated in the presence or absence of 0.5 mM NaHS in the darkness for 2 d. (e), Photographs of detached wild type leaves treated with H₂S released from 0.5 mM NaHS solution under extended darkness for 2 d and transfer to regular growth conditions for another 2 d. Data are means ± SE of at least three independent samples from different plants. Asterisks indicate significant difference between H₂S-treated wild type and control wild type at the same time point at P < 0.05, using the Student’s t test.

Figure 3

Effects of H₂S exposure on phenotype of whole plants under prolonged darkness and regular growth conditions. Photographs of whole plants treated with different NaHS concentrations exposed to complete darkness for 2 d (a) and then transfer to regular growth conditions for another 1 d (b) (16 h light/8 h dark photoperiod). The fresh weight of plants treated with the indicated concentrations of NaHS was measured 6 d after transfer from continuous darkness to light-dark conditions (c). (d), Effects of 0.5 mM NaHS treatment under prolonged darkness or regular 16 h light/8 h dark conditions for 3 days. (e) Fresh weight were taken at 7 d after H₂S treatment under 16 h light/8 h dark conditions. Data are means ± SE of at least 15 different plants. ND: not detected.

Figure 4

Effects of exogenously applied H₂S and DES1 transgenic lines on SAG expression in attached leaves under extended darkness. Transcript levels of SAG12 (a), SAG20 (b) and SEN4 (c) in wild-type plants subjected to 0.5 mM NaHS treatment plus complete darkness. Samples were taken from the attached leaves at 2 or 4 d of darkness. + and − indicate intact plants fumigated with or without 0.5 mM NaHS, respectively. (d), SAG12. (e), SAG20. (f), SEN4. Samples were taken from the attached leaves at 2 and 4 d of darkness. OE1 and OE2 indicate two independent DES1 overexpression lines. Data are means ± SE of at least three independent samples from different plants. Asterisks indicate significant difference from the wild type at the same time point at P < 0.05, using the Student’s t test.

Figure 5

Expressions of of SAGs and PR1 gene in attached leaves of Col-0 and des1 mutant during extended darkness. (a), SAG12. (b), SAG20. (c), SEN4. (d), PR1. Ten-day-old seedlings of Col-0 and des1 were incubated under extended darkness for up to 8 d. White bars, des1 mutant. Black bars, Col-0. Asterisks indicate significant difference from the wild type at the same time point at P < 0.05, using the Student’s t test.

Figure 6

Effects of H₂S exposure on leaf H₂O₂, glutathione and ascorbate in attached leaves of Col-0 under extended darkness and normal growth conditions. (a), H₂O₂ content. Samples were taken from the attached leaves at 2 and 4 d of darkness. (b), reduced glutathione (white bars) and oxidized glutathione (black bars). (c), ascorbate (white bars) and dehydroascorbate (black bars). Samples were taken from the attached leaves at 2 d of darkness and regular growth conditions within 16 h light/8 h dark photoperiod. + and − indicate intact plants fumigated with or without 0.5 mM NaHS, respectively. Light indicates regular growth conditions within 16 h light/8 h dark photoperiod. Data are means ± SE of at least three independent samples from different plants. Letters indicates significant difference from the wild type at P < 0.05, using the Student’s t test.

Figure 7

Effects of H₂S exposure on major antioxidative enzyme in attached leaves of Col-0 under extended darkness and normal growth conditions. (a) APX. (b), CAT. (c), DHAR. (d), GR. Samples were taken from the attached leaves at 2 d of darkness and light/dark growth conditions. + and − indicate intact plants fumigated with or without 0.5 mM NaHS, respectively, during 2 d of dark incubation and light/dark growth conditions. Data are means ± SE of at least three independent samples from different plants. Letters indicates significant difference from the wild type at P < 0.05, using the Student’s t test.

Figure 8

SAG expression in attached leaves of Col-0 and gsnor1 treated with or without H₂S under extended darkness. (a), SAG12 expression. (b), SAG20 expression. (c), SEN4 expression. Samples were taken from the attached leaves at 4 d of darkness treatment. + and − indicate intact plants fumigated with or without 0.5 mM NaHS, respectively, during 4 d of dark incubation. Data are means ± SE of at least three independent samples from different plants. Letters indicates significant difference from the wild type at P < 0.05, using the Student’s t test.