Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance

Abstract

Background and aims: Rice is one of the few crops able to withstand periods of partial or even complete submergence. One of the adaptive traits of rice is the constitutive presence and further development of aerenchyma which enables oxygen to be transported to submerged organs. The development of lysigenous aerenchyma is promoted by ethylene accumulating within the submerged plant tissues, although other signalling mechanisms may also co-exist. In this study, aerenchyma development was analysed in two rice (Oryza sativa) varieties, 'FR13A' and 'Arborio Precoce', which show opposite traits in flooding response in terms of internode elongation and survival.

Methods: The growth and survival of rice varieties under submergence was investigated in the leaf sheath of 'FR13A' and 'Arborio Precoce'. The possible involvement of ethylene and reactive oxygen species (ROS) was evaluated in relation to aerenchyma formation. Cell viability and DNA fragmentation were determined by FDA/FM4-64 staining and TUNEL assay, respectively. Ethylene production was monitored by gas chromatography and by analysing ACO gene expression. ROS production was measured by using Amplex Red assay kit and the fluorescent dye DCFH(2)-DA. The expression of APX1 was also evaluated. AVG and DPI solutions were used to test the effect of inhibiting ethylene biosynthesis and ROS production, respectively.

Key results: Both the varieties displayed constitutive lysigenous aerenchyma formation, which was further enhanced when submerged. 'Arborio Precoce', which is characterized by fast elongation when submerged, showed active ethylene biosynthetic machinery associated with increased aerenchymatous areas. 'FR13A', which harbours the Sub1A gene that limits growth during oxygen deprivation, did not show any increase in ethylene production after submersion but still displayed increased aerenchyma. Hydrogen peroxide levels increased in 'FR13A' but not in 'Arborio Precoce'.

Conclusions: While ethylene controls aerenchyma formation in the fast-elongating 'Arborio Precoce' variety, in 'FR13A' ROS accumulation plays an important role.

Fig. 1.

‘Arborio Precoce’ (‘AP’) and ‘FR13A’ plants under submergence. (A) Diagram showing how the rice varieties were submerged in water. One-week-old rice seedlings were grown in pots and flooded with water, 2 cm (minimal), 15 cm (partial) or 30 cm (total) above soil surface. The drawing depicts the plants at the end of the experiment. (B) Shoot length of the rice plants under different submergence conditions. The blue lines indicate the water level. Data are expressed as mean ± s.d., n = 12. (C) Percentage of plant survival after 21 d of submersion followed by 7 d of recovery under well-drained conditions. Data are expressed as mean ± s.e., n = 12; **, P < 0·01according to Student's t-test.

Fig. 2.

Aerenchyma formation in leaf sheath of ‘AP’ and ‘FR13A’ under air and after 3 d of total plant submergence (Sub). (A) Representative fresh cross-sections of the leaf sheath rice varieties under air and submergence. (B) Percentage of aerenchymatous area in leaf sheath sections of the two varieties. Data are expressed as mean ± s.e., n = 9; * P < 0·05 and ** P < 0·01 according to Student's t-test. (C) Viability of ‘AP’ and ‘FR13A’ leaf sheath cells showing aerenchyma formation using double staining with FM4-64 and FDA. Viable cells stained green by FDA indicate intact plasma membranes. Absence of green colour and substantial red cell staining with FM4-64 indicates the loss of membrane integrity (arrow). Abbreviations: AE, aerenchyma; SC, schlerenchyma. Scale bar = 0·2 mm.

Fig. 3.

DNA fragmentation in ‘AP’ and ‘FR13A’ leaf sheaths under air and after 3 d of total plant submergence (sub), detected by TUNEL staining. DAPI-stained nuclei were used as positive controls. Scale bar = 0·2 mm.

Fig. 4.

Ethylene synthesis in ‘AP’ and ‘FR13A’ leaf sheaths during aerenchyma formation. (A) Expression of Sub1A and ACO genes. The expression level was measured based on ‘FR13A’ air control at day 0 = 1. Data are mean ± s.d., n = 3. *** P < 0·001, ** P < 0·01 and * P < 0·05 according to Student's t-test. (B) Ethylene production after 3 d air and after 3 d of submergence treatment. Data are mean ± s.d., n = 3. ** P < 0·01 according to Student's t-test. (C) Percentage of aerenchymatous area in leaf sheath sections of ‘AP’ and ‘FR13A’ after the submergence treatment, with or without the ethylene biosynthesis inhibitor AVG (500 µm), every 24 h for 3 d before and during submergence. Data are mean ± s.e., n = 9. Different lower-case letters indicate significant differences between treatments (0·05 significance level) based on LSD multiple pairwise comparison test.

Fig. 5.

H₂O₂ production in ‘AP’ and ‘FR13A’ leaf sheaths. (A) H₂O₂ production after 3 d of submergence treatment and in well-drained conditions (air). Data are the mean ± s.d., n = 3; * P < 0·05 according to Student's t-test. (B) Expression pattern of APX1. The expression level was measured based on ‘FR13A’ control air at day 0 = 1. Data are the mean ± s.d., n = 3; ** P < 0·01 and * P < 0·05 according to Student's t-test. (C) Leaf sheath sections of ‘AP’ and ‘FR13A’ treated with the green fluorescent dye H₂-DCFDA for the detection of ROS. Scale bar = 0·2 mm. (D) Percentage of aerenchymatous area in leaf sheath sections of ‘AP’ and ‘FR13A’ after the submergence treatment, with or without DPI (10 µm), every 24 h for 3 d before and during submergence. Data are mean ± s.e., n = 3. Different lower-case letters indicate significant differences between treatments (0·05 significance level) based on LSD multiple pairwise comparison.