Ante- and post-mortem cellular injury dynamics in hybrid poplar foliage as a function of phytotoxic O3 dose

Abstract

After reaching phytotoxic levels during the last century, tropospheric ozone (O3) pollution is likely to remain a major concern in the coming decades. Despite similar injury processes, there is astounding interspecific-and sometimes intraspecific-foliar symptom variability, which may be related to spatial and temporal variation in injury dynamics. After characterizing the dynamics of physiological responses and O3 injury in the foliage of hybrid poplar in an earlier study, here we investigated the dynamics of changes in the cell structure occurring in the mesophyll as a function of O3 treatment, time, phytotoxic O3 dose (POD0), leaf developmental stage, and mesophyll layer. While the number of Hypersensitive Response-like (HR-like) lesions increased with higher O3 concentrations and POD0, especially in older leaves, most structural HR-like markers developed after cell death, independent of the experimental factors. The pace of degenerative Accelerated Cell Senescence (ACS) responses depended closely on the O3 concentration and POD0, in interaction with leaf age. Changes in total chlorophyll content, plastoglobuli and chloroplast shape pointed to thylakoid membranes in chloroplasts as being especially sensitive to O3 stress. Hence, our study demonstrates that early HR-like markers can provide reasonably specific, sensitive and reliable quantitative structural estimates of O3 stress for e.g. risk assessment studies, especially if they are associated with degenerative and thylakoid-related injury in chloroplasts from mesophyll.

Fig 1. Development dynamics of non-oxidized and oxidized HR-like lesions at the 3^rd leaf position in hybrid poplar (Populus tremula x alba), as a function of O₃ treatment and time.

{model: lmer[log(variable+1) ~ leaf position * Oxidation * Time * O₃ treatment + (1|pot)]; *** P ≤ 0.001; * P ≤ 0.05}. The inset image is a synthetic image of the particle distribution and morphology in each lesion color class (non-oxidized/oxidized) during image analyses of HR-like reactions. Values represent percentage area means ± SE of leaf discs showing non-oxidized or oxidized HR-like lesions (n = 4). Different letters indicate significant differences between treatments at a given assessment date (Tukey’s honestly significant difference (HSD) post-hoc test, P ≤ 0.05).

Fig 2. Dynamics of changes in the total chlorophyll concentration within leaves of hybrid poplar (Populus tremula x alba) as a function of assessment time (A; mg g^-1 of fresh matter), POD₀ (B; percentage of values in the CF treatment).

O₃ treatment (dotted line: CF, dashed line: CF +80 ppb O₃, long dash line: CF +100 ppb O₃), leaf position, and their interactions (model: lmer(variable) ~ O₃ treatment * leaf position * time or POD₀ + (1 | tree/chamber)); *** P ≤ 0.001, * P ≤ 0.05, ns not significantly different). Values represent means ± SE (n = 4). Different letters indicate significant differences between treatments on a given assessment date (post-hoc Tukey’s HSD, P ≤ 0.05).

Fig 3

Evolution of cellular and subcellular traits in HR-like lesions within mesophyll cells of hybrid poplar (Populus tremula x alba) leaves at the 3^rd (A–D, K–P) and 10^th leaf position (E–J) and in response to the CF+100 ppb O₃ treatment. A–D Asymptomatic mesophyll cell structure in the CF treatment. E–F Early stage of HR-like reaction development: cell death and cytorrhysis. In apparent relation to cell cytorrhysis, the vacuole was shrunken (sv) and its irregular shape indicated tonoplast rupture, whereas other organelles were still intact. G–K Intermediate stage of HR-like reaction development: disruption of cell content. H Within mitochondria, degradation of the mitochondrial matrix (!) and disruption of outer membrane (arrow). I Nucleus pyknosis (pn) and condensation of cytoplasm (~). J chloroplast (ch) disruption. K–K’ Leakage of cell debris (cm) into the intercellular space (is) through break points in the cell wall (#). L–N Final stage of HR-like reaction development: condensation and fusion of cellular debris. O–P Local thickening of cell wall (cw) through callose deposition within cells adjacent to lesions. Other structures: g: grana, n: nucleus, v: vacuole. Technical specifications: post-fixation using OsO4; contrasting using uranyl acetate and lead citrate; observation in TEM (A, B–E, F, G, H-J, L, M, N, P); staining with Toluidine blue and observation under phase contrast in bright field microscopy (A’, E’, G’-K’, L’); staining with Aniline blue and observation with fluorescence microscopy (O).

Fig 4

Evolution of degenerative traits at the cellular and subcellular level within mesophyll cells surrounding HR-like lesions inside of hybrid poplar (Populus tremula x alba) leaves at the 3^rd leaf position and in response to the CF+100 ppb O₃ (K–O, P–T) treatment relative to the CF treatment (A–E, F–J). Treatment time: 8 days (A–E, K–O) or 23 days (F–J, P–T). The main difference between the two treatments was the quicker evolution of degenerative traits in the O₃-treated trees (F, G) relative to the CF trees (P, Q). Markers at the cellular level: relative increase in vacuole (v) size at the expense of cytoplasm and the apparent reduction in the number and size of chloroplasts (ch). Markers at the subcellular level: within chloroplasts: increase in starch grain (st) accumulation (K–M vs. A–C), injury to thylakoid membranes of grana (g; R, T vs. H, J) and increase in the plastoglobuli (pg) size and frequency (Q, R, vs. G, H); within nucleus (n): condensation of chromatin and loss of nucleoli (nl; N, S vs. D, I); in the vacuole: accumulation of secondary metabolites (sp; P vs. F). Other structures: cw: cell wall, is: intercellular space. Technical specifications: post-fixation using OsO₄, contrasting using uranyl acetate and lead citrate, observation in TEM (B–E, G–J, L–O, Q–T); staining with Toluidine blue, observation under phase contrast in bright field microscopy (A, F, K, P).

Fig 5. Contrast in the cell wall thickening processes at the 3^rd (A–C) vs. 10^th leaf position (D–G) within leaf mesophyll cells undergoing ACS processes from hybrid poplar (Populus tremula x alba) trees in the CF+100 ppb O₃ treatment.

A–C At the 3^rd leaf position, cell wall thickening primarily resulted from exocytosis (~) of multivesicular body (mvb) content into the periplasm (pe), as a consequence of autophagic processes. Notice the thin cytoplasm (ct) layer and few organelles. D–G At the 10^th leaf position, ontological cell wall thickening in response to treatment was indicated by a dense network of mitochondria (mt; D), rough endoplasmic reticulum (rer; F), and dictyosomes (d; E) secreting vesicles (arrowheads in E, G) heading towards apoplast. Other structures: ch: chloroplast, cw: cell wall, is: intercellular space, v: vacuole. Technical specifications: post-fixation using OsO₄, contrasting using uranyl acetate and lead citrate, observation in TEM.

Fig 6. Visualization of chloroplast size and shape trait dynamics as a function of O₃ treatment, leaf position, assessment time or POD₀, mesophyll layer, and interactions, as observed in the leaf mesophyll of hybrid poplar trees (Populus tremula x alba) on the 8^th, 13^th and 23^rd day of treatment.

Model: lmer(variable ~ O₃ treatment * leaf position * time + (1 | cell/tree)); *** P ≤ 0.001, * P ≤ 0.05, ns not significantly different). Data basis for the conceptual models of chloroplast trait changes: chloroplast long and short axis (green ellipse), cumulated starch grain and plastoglobuli percentage area (blue and red circles), total chlorophyll content (in mg g^-1 FM; green color scale).