Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Abstract

Background and aims: Plants regulate cellular oxygen partial pressures (pO2), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO2 regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.

Methods: The gradients in pO2 from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO2 production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.

Key results: Steep internal pO2 gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO2 production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO2 gradient showed a biphasic profile, where the minimum pO2 was external to the core of the bud complex.

Conclusions: Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.

Keywords: Bud burst; ROS; Vitis vinifera; development; ecodormancy; grapevine; hypoxia; meristem; oxygen partial pressure; quiescence; reactive oxygen species; respiration; superoxide.

Fig. 1.

Time-series of grapevine bud burst. Single node explants of ecodormant buds were transferred from cool storage (4 °C) and planted out at 23 °C (dark). Figure shows the progression of bud burst at 0, 1, 3, 7 and 9 d (left to right) at 23 °C. Buds were sampled for the studies presented here at select time points during this development. The inset shows a sagittal section of the bud, with the primary (centre arrow), secondary (right arrow) and tertiary (left arrow) bud meristem complexes. When ecodormant (0 d), the bud complex is enclosed by a layer of lignified scales and several layers of bracts. Progressively over 3–5 d we observed expansion of the bud complex and rupture of the outer scales. Within 5–7 d, buds reached the stage of bud burst, according to the modified Eichorn–Lorenz scale (EL4; Coombe, 2004). By 9 d, the first leaves had separated from the shoot apical meristem (EL7). Scale bar in main figure = 5 mm, inset = 1 mm.

Fig. 2.

Respiratory CO₂ production during grapevine bud burst. Ecodormant buds were transferred from cool storage (4 °C) and planted out at 23 °C (dark) at 0 h. The rate of CO₂ production was measured on groups of four excised buds with the cut base on agar using an infra-red gas analyser in darkness. Data represent a regression (n = 4 replicates of four buds per replicate) ± 95 % confidence intervals by fitting the time series of CO₂ evolution to a quadratic equation of the form, y = α + β₁x + β₂x² (refer Materials and Methods).

Fig. 3.

Internal profile of the partial pressure of oxygen (pO₂) during grapevine bud burst. The pO₂ of ecodormant buds, intact (A = 3 h, C = 24 h, D = 72 h) or with the outer scale removed (B = 3 h) was assayed after time at 23 °C in darkness. Data represent scatterplots of raw data (n = 3), with a regression curve applied and 95 % confidence intervals shown as grey shading. (E) Sagittal section of the primary bud meristem complex, fixed and stained with toluidine blue, showing the path of the O₂ microelectrode from the outer scale (arrow) towards the inner core of the primary bud complex. Scale bar = 500 µm.

Fig. 4.

Spatial and temporal localization of reactive oxygen species (ROS) in sagittal sections of the primary bud meristem complex during bud burst. Superoxide (A–D) and hydrogen peroxide (E–H) localization were indicated using nitrobluetetrazolium (NBT) and 3,3′diaminobenzedine (DAB), respectively, against fixed sections (20 µm), sampled at 0 h (A, E), 3 h (B, F), 24 h (C, G) or 72 h (D, H) after transfer to 23 °C. Scale bar = 500 µm. Figures are representative of three independent replicates.

Fig. 5.

Spatial and temporal localization of superoxide (A, C, E) as contrasted to lignin (B, D, F) in grapevine buds during the first 3 h after transfer to 23 °C. Superoxide (NBT) is localized to latent meristem cells at 0 h, with negligible association with lignified cells (A–D, indicated by Auramine-O), where C and D are magnifications of the boxed inserts in A and B. By 3 h at 23 °C, superoxide production is evidently associated with lignin, indicative of pro-vascular development (E–G), where G is F superimposed over E. Scale bar = 100 µm (A, B), 20 µm (C, D), 50 µm (E–G). Figures are representative of three independent replicates.