The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress in barley (Hordeum vulgare L.)

Abstract

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression. Comparison of wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1 showed, that the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress. The results support a central role of WHIRLY1 in retrograde signaling and also underpin a so far underestimated role of microRNAs in this process.

Keywords: Retrograde signaling; small RNA; stress response.

Figure 1.

Characterization of WHIRLY1 knockdown lines at the seedling stage. Seedlings were exposed to continuous irradiation at 50, 120, 200 or 350 µmol photons m⁻² s⁻¹ for 7 days. Lengths of the primary leaves (cm) are indicated (a). Pigment extracts from the wild type (WT) and the RNAi-W1 lines (W1-1, W1-7, W1-9) were compared by HPLC for the content of chlorophylls/leaf area (b), the ratio of xanthophyll cycle pigments (VAZ) to chlorophyll (c) and the de-epoxidation state of VAZ (d). De-epoxidation state was calculated as (Z + 0.5A)/(V + A + Z). All data are means of 3 samples, error bars denote standard deviation. The results obtained for lines RNAi-W1-1 and RNAi-W1-9 are rather similar. Only the results of RNAi-W1-1 are therefore shown.

Figure 2.

ROS production by thylakoids from wild type and RNAi-W1-1 and RNAi-W1-7 lines. Thylakoids were prepared from seedlings grown in continuous light of 200 µmol photons m⁻¹s⁻¹. Superoxide/hydrogen peroxide levels were measured by spin trapping EPR using 4-POBN/EtOH/FeEDTA as spintrap and singlet oxygen by the spin probe TEMPD-HCl (for experimental details, see [15]). Thylakoids were illuminated for two minutes with red light (500 µmol quanta m⁻²s⁻¹) in the presence of the chemicals. Left: representative spectra, right: EPR signal sizes (4-POBN/EtOH/FeEDTA) were normalized to the signal obtained in wild-type thylakoids (mean ± SD, n = 6).

Figure 3.

RT-qPCR analysis of microRNAs and expression of target genes in wild type (WT) and transgenic RNAi -W1-7 plants exposed to either low (LL) or high light (HL). (a) In WT plants exposed to HL the levels of microRNAs were enhanced. Results are presented as fold changes and results for WT plants grown in LL are treated as 1. (b) In the wild type plants HL lead to a downregulation of the levels of target mRNAs. (c) Levels of most target mRNAs were enhanced in HL treated W1-7 plants when compared to the WT. (d) Target mRNAs expression stayed mostly unchanged when RNAi-W1-7 plants exposed to LL and HL are compared. Error bars indicate SD (n = 3), and the asterisk indicates a significant difference between the sample and control (t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).