Interaction Between Zn Deficiency, Toxicity and Turnip Yellow Mosaic Virus Infection in Noccaea ochroleucum

Abstract

Zinc is essential for the functioning of numerous proteins in plants. To investigate how Zn homeostasis interacts with virus infection, Zn-tolerant Noccaea ochroleucum plants exposed to deficient (Zn'0'), optimal (Zn10), and excess Zn (Zn100) concentrations, as well as Cd amendment, were infected with Turnip yellow mosaic virus (TYMV). Imaging analysis of fluorescence kinetics from the μs (OJIP) to the minutes (Kautsky effect, quenching analysis) time domain revealed strong patchiness of systemic virus-induced photosystem II (PSII) inhibition. That was more pronounced in Zn-deficient plants, while Zn excess acted synergistically with TYMV, in both cases resulting in reduced PSII reaction centers. Infected Cd-treated plants, already severely stressed, showed inhibited non-photochemical quenching and PSII activity. Quantitative in situ hybridization at the cellular level showed increased gene expression of ZNT5 and downregulation of HMA4 in infected Zn-deficient leaves. In Zn10 and Zn100 infected leaves, vacuolar sequestration of Zn increased by activation of HMA3 (mesophyll) and MTP1 (epidermis). This correlated with Zn accumulation in the mesophyll and formation of biomineralization dots in the cell wall (Zn100) visible by micro X-ray fluorescence tomography. The study reveals the importance of adequate Zn supply and distribution in the maintenance of photosynthesis under TYMV infection, achieved by tissue-targeted activation of metal transporter gene expression.

Keywords: TYMV infection; cadmium; chlorophyll fluorescence kinetics; metal transporters; micro X-ray fluorescence; non-hyperaccumulator; plant immunity; zinc.

FIGURE 1

OJIP parameters. Zn0, Zn10, and Zn100 refer to Zn concentrations used in the treatments (‘0’ = 0.01, 10 and 100 μM Zn), N-non-infected – empty box plots. I-infected- box plots with stripes. Cd1 refers to a 1 μM Cd + 10 μM Zn treatment. The line presents the median value (n = 6–8), the box shows the values between the 0.25–0.75 percentiles, and the bars show whiskers with 1.5 coefficient for outliers. Asterisks denote significant differences between infected and non-infected N. ochroleucum plants within the same Zn treatment according to the Mann–Whitney test. (A) Φ _Po, maximum quantum yield of primary PSII photochemistry, (B) Φ _ET2o, quantum yield of electron transport flux from Q _A to Q_B; (C) Φ_RE1o, quantum yield of electron transport flux until PSI acceptors; (D) J^abs/RC, average absorbed photon flux per PSII reaction center, (E) J_o^ET2/RC, electron transport flux from Q _A to Q_B per PSII; (F) J_o^Re1o/RC, electron transport flux until PSI acceptors according to Stirbet and Govindjee (2011).

FIGURE 2

Operating PSII efficiency in the irradiation phase, with the dark-adapted maximal quantum yield of PSIRC (F_v/F _m) as a reference. Zn0 (A), Zn10 (B), and Zn100 (C) refer to Zn concentrations used in the treatments (‘0’ = 0.01, 10 and 100 μM Zn), Cd1 (D) refers to a 1 μM Cd + 10 μM Zn treatment. The line presents the median value (n = 6–8), the box shows the values between the (0.25–0.75 percentiles, and the bars show whiskers with 1.5 coefficient for outliers. Asterisks denote significant differences between infected and non-infected N. ochroleucum plants within the same Zn treatment according to the Mann–Whitney test. F _m, maximal chlorophyll fluorescence in dark-adapted state; F _m’, maximal chlorophyll fluorescence in light-adapted state; F₀, minimal chlorophyll fluorescence in dark-adapted state; F_v, variable fluorescence = (F _m-F₀)/F _m; Φ _PSII = (F _m’-F_t’)/F _m’) from the beginning (i1) to the end (i6) of the 200 s actinic light phase using 600 ms flash of saturating light.)

FIGURE 3

Complete NPQ ((F _m-F _m’)/F _m) during the irradiance phase. Zn0 (A), Zn10 (B) and Zn100 (C) refer to Zn concentrations used in the treatments (‘0’ = 0.01, 10 and 100 μM Zn), Cd1 (D) refers to a 1 μM Cd + 10 μM Zn treatment. The line presents the median value (n = 6–8), the box shows the values between the 0.25–0.75 percentiles, and the bars show whiskers with 1.5 coefficient for outliers. Asterisks denote significant differences between infected and non-infected N. ochroleucum plants within the same Zn treatment according to the Mann–Whitney test. F _m, maximal chlorophyll fluorescence in dark-adapted state; F _m’, maximal chlorophyll fluorescence in light-adapted state; NPQ was measured from the beginning (i1) to the end (i6) of the 200 s actinic light phase using 600 ms flashes of saturating light.

FIGURE 4

Expression of metal transporter genes normalized to cytosolic GAPDH in the leaves of N. ochroleucum plants calculated according to Pfaffl. Zn0 (A), Zn10 (B) and Zn100 (C) refer to Zn concentrations used in the treatments (‘0’ = 0.01, 10 and 100 μM Zn), Cd1 (D) refers to a 1 μM Cd + 10 μM Zn treatment. N – non-infected – empty box plots. I – infected – box plots with stripes. The line presents the median value (n = 6–8), the box shows the values between the 0.25–0.75 percentiles, and the bars show whiskers with 1.5 coefficient for outliers. Asterisks denote significant differences between infected and non-infected plants within the same Zn treatment according to the Mann–Whitney test.

FIGURE 5

Expression of metal transporter genes normalized to 18S in the leaves of N. ochroleucum plants calculated according to Pfaffl. Zn0 (A), Zn10 (B) and Zn100 (C) refer to Zn concentrations used in the treatments (‘0’ = 0.01, 10 and 100 μM Zn), Cd1 (D) refers to 1 μM Cd + 10 μM Zn treatment. N – non-infected – empty box plots. I – infected – box plots with stripes. The line presents the median value (n = 6–8), the box shows the values between the 0.25–0.75 percentiles, and the bars show whiskers with 1.5 coefficient for outliers. Asterisks denote significant differences between infected and non-infected plants within the same Zn treatment according to the Mann–Whitney test.

FIGURE 6

Images showing the gene expression ratio in different tissues of metal transporters to cGADPH probe fluorescent signals of TYMV infected and non-infected N. ochroleucum leaves. Zn0, Zn10, and Zn100 refer to Zn concentrations used in the treatments (‘0’ = 0.01, 10 and 100 μM Zn), Cd1 refers to a 1 μM Cd + 10 μM Zn treatment. The scale bars for all genes within the same tissue are shown.

FIGURE 7

μXRF tomography of Zn distribution in fully developed N. ochroleucum leaves. The samples were taken at the time of harvest of the plants. At least two replicates from independent experiments were measured for each panel of this figure; representative examples are shown. Top: plants grown with 10 μM Zn²⁺; bottom: 100 μM Zn²⁺; Left: non-infected plants; right: TYMV-infected plants. The scale bar in the top-right map is valid for all four maps.