Rice plants overexpressing OsEPF1 show reduced stomatal density and increased root cortical aerenchyma formation

Abstract

Stomata are adjustable pores in the aerial epidermis of plants. The role of stomata is usually described in terms of the trade-off between CO₂ uptake and water loss. Little consideration has been given to their interaction with below-ground development or diffusion of other gases. We overexpressed the rice EPIDERMAL PATTERNING FACTOR1 (OsEPF1) to produce rice plants with reduced stomatal densities, resulting in lowered leaf stomatal conductance and enhanced water use efficiency. Surprisingly, we found that root cortical aerenchyma (RCA) is formed constitutively in OsEPF1OE lines regardless of tissue age and position. Aerenchyma is tissue containing air-spaces that can develop in the plant root during stressful conditions, e.g. oxygen deficiency when it functions to increase O₂ diffusion from shoot to root. The relationship with stomata is unknown. We conclude that RCA development and stomatal development are linked by two possible mechanisms: first that reduced stomatal conductance inhibits the diffusion of oxygen to the root, creating an oxygen deficit and stimulating the formation of RCA, second that an unknown EPF signalling pathway may be involved. Our observations have fundamental implications for the understanding of whole plant gas diffusion and root-to-shoot signalling events.

Figure 1

Overexpression of OsEPF/L genes in rice causes alterations in stomatal size and density. (a) Expression analysis showing overexpression of OsEPF1 and OsEPFL9 genes in independent transgenic lines, ai. Reverse transcription – PCR of OsEPF1OE (line 3 and line 4) and OsEPFL9OE (Line 1 and 4), L denotes ladder, aii. Quantitative RT-PCR of OsEPF1OE (line 3 and 4) and OsEPFL9OE (Line 1, 15 and 17). (b,c) Stomatal density (b) and stomatal size (c) measurements of 5 week old OsEPF1 and OsEPFL9 overexpression lines on the adaxial and abaxial leaf surfaces of a fully developed 5^th leaf. (d) The relationship between the mean of the stomatal sizes and mean of the stomatal densities of the adaxial (closed circles) and abaxial (open circles) surfaces. (e,f) Leaf impressions showing stomata distribution in untransformed wildtype (WT) controls and OsEPF overexpression lines on adaxial (e) and abaxial (f) surfaces. TN refers to (transformed) transgene negative plants. Data are represented as mean ± SEM. n = 4–6. Different letters indicate significant differences between the lines. (p < 0.05). Scale bar = 25 µm.

Figure 2

OsEPF overexpression lines show altered stomatal conductance and comparable photosynthesis to control plants. Five week old control plants and OsEPF overexpression lines were used for the analysis of (a) Stomatal conductance (gs), (b) CO₂ assimilation rate (A), (c) Transpiration rate (E), (d) Leaf temperature, (e) Instantaneous water use efficiency (A/E), (f) Intrinsic water use efficiency (A/gs). All values are means ± SE (n = 4–5, Letters represent significant differences among the genotypes. P ≤ 0.05).

Figure 3

Reduced stomatal density slows leaf dehydration. Rapid leaf dehydration assay (leaf excision) of youngest fully expanded leaf on 8 week old plants. (a) The rate of water loss over time in an excised leaf (excised at 0 seconds). (b) Stomatal conductance in an excised leaf. The values were normalized to the initial value just prior to excision. Each data point represented as the mean and error bars indicate ± SEM. The asterisks represent the significant differences between the OsEPF1OE and the WT (*P ≤ 0.05 and ****P ≤ 0.0001). N = 4–6.

Figure 4

Reduced stomatal density improves soil water conservation. (a) Changes in soil water content in 5 week old plants over 8 days of drought stress. (b) Mean leaf temperatures during the drought stress and following re-watering compared to soil water at saturated state. Each data point represented as the mean and error bars indicate ± SEM. The asterisks represent the significant differences between the OsEPF1OE and the WT (*P ≤ 0.05). N = 4–5.

Figure 5

Reduced stomatal density improves leaf turgor and water conservation. OsEPF1OE plants show better leaf turgor and water retention than the WT leaves. (a–d) Representative 5 week (a,b) and 8 week (c,d) old (post germination) WT and OsEPF1OE plants on day 6 of drought stress. (e,f) Relative water content of 5 week (e) and 8 week (f) old (post germination) plants at day 6 of drought stress, normalised to the well-watered plants. The asterisk (*) represents the significant differences between the two treatments (*P ≤ 0.05 and **P ≤ 0.01) and error bars indicate SEM. Scale bar = 12 cm.

Figure 6

Overexpression of OsEPF1 results in an increase in root cortical aerenchyma. Spatiotemporal formation of aerenchyma in 60-day old plants induced by constitutive overexpression of OsEPF1 and OsEPFL9. (a) RCA was spatiotemporally evaluated in two positions; 2 cm and 13 cm from the roots tips. (b–g) Sections taken at 13 cm (b–d) and at 2 cm (e–g) of WT, OsEPF1OE and OsEPFL9OE roots. (h) Estimated aerenchyma formed in the two root regions. Different letters above the columns indicate significant differences between lines and sampling region (p < 0.05). All values are represented as the mean ± SEM. N = 4–7. Scale bar = 50 µm.

Figure 7

Dissolved Oxygen levels impact aerenchyma formation. The adventitious root section of the 35-day old plant roots were sampled at 6 cm from the root tip. a-r. The RCA formation (a–c,g–i,m–o) and estimation of aerenchyma via Rootscan 2 (d–f,j–l,p–r). WT (a–f), OsEPF1OE (g–l) and OsEPFL9OE (m–r) roots at three different dissolved oxygen levels (95%, 66%, and 28%). (s) Bar graph showing estimated RCA at varying D.O levels for WT, two OsEPF1OE lines and two OsEPFL9OE lines. Different letters indicate significant differences between the lines (p < 0.05). All values are represented as the mean ± SEM. N = 8. Scale bar = 50 µm.

Figure 8

Stomatal density is quantitatively related to root aerenchyma formation. The linear regression of 5-week old plants between the estimated aerenchyma at 95% D.O level and the abaxial stomatal density of the WT, OsEPF1OE, and OsEPFL9OE in aerated condition. (P = 0.0001 and R² = 0.3020). N = 7–12.