Isochorismate synthase 1 is required for thylakoid organization, optimal plastoquinone redox status, and state transitions in Arabidopsis thaliana

Abstract

Isochorismate synthase 1 (ICS1) is a crucial enzyme in the salicylic acid (SA) synthesis pathway, and thus it is important for immune defences. The ics1 mutant is used in experiments on plant-pathogen interactions, and ICS1 is required for the appropriate hypersensitive disease defence response. However, ICS1 also takes part in the synthesis of phylloquinone, which is incorporated into photosystem I and is an important component of photosynthetic electron transport in plants. Therefore, photosynthetic and molecular analysis of the ics1 mutant in comparison with wild-type and SA-degrading transgenic NahG Arabidopsis thaliana plants was performed. Photosynthetic parameters in the ics1 mutant, when compared with the wild type, were changed in a manner observed previously for state transition-impaired plants (STN7 kinase recessive mutant, stn7). In contrast to stn7, deregulation of the redox status of the plastoquinone pool (measured as 1-q p) in ics1 showed significant variation depending on the leaf age. SA-degrading transgenic NahG plants targeted to the cytoplasm or chloroplasts displayed normal (wild-type-like) state transition. However, ics1 plants treated with a phylloquinone precursor displayed symptoms of phenotypic reversion towards the wild type. ics1 also showed altered thylakoid structure with an increased number of stacked thylakoids per granum which indicates the role of ICS1 in regulation of state transition. The results presented here suggest the role of ICS1 in integration of the chloroplast ultrastructure, the redox status of the plastoquinone pool, and organization of the photosystems, which all are important for optimal immune defence and light acclimatory responses.

Keywords: Chlorophyll fluorescence; photosynthetic electron transport (PET); phylloquinone; plastoquinone pool (PQ pool); salicylic acid (SA); state transitions (ST)..

Fig. 1.

State transition in wild-type and ics1 plants (a), NahG transgenic plants with both a cytosolic- and chloroplast-targeted SA-degrading enzyme (b), and wild-type and ics1 plants supplemented with the phylloquinone precursor (1,4-dihydroxy-2-naphthoic acid; NA) (c). The top bar depicts lights applied during a particular period of the measurement; blue, actinic light on; red, far-red light on. The graph shows representative measurements of six whole rosettes for each genotype and treatment. The inset in (c) shows the q _S parameter (mean ±SD) for the analysed genotypes and treatments. Homogeneous groups were calculated using Tukey HSD analysis. For details, see the Materials and methods.

Fig. 2.

Representative pictures of the chloroplast structure from WT and ics1 plants. The WT chloroplasts show a typical structure with a lamellar system of granal and stromal thylakoids, while in ics1 enlargement of the granum showing numerous stacked thylakoid sheets can be observed. CW, cell wall; GT, grana thylakoids; PG, plastoglobuli; S, starch; ST, stroma thylakoids; Str, stroma; V, vacuole. Bar=2 μm.

Fig. 3.

Redox status of the plastoquinone pool. (a) Light curve of the redox status of the PQ pool (1–q _P). Data represent an average from 10 different rosettes (n=10, grey shading indicates the 95% confidence intervals for the analysed genotypes). (b and c) Thermoluminescence (TL) measurements. (b) TL glow curves in wild-type (WT, red dots) and ics1 (blue dots) plants. The figure shows representative measurement of the leaves excited with two flashes. (c) Flash-induced oscillation of the B-band amplitude (TL_max) in wild-type (WT, red line and dots) and ics1 (blue line and triangles) plants. Values are means ±SEM of seven independent measurements (leaves from seven independent plants). Asterisks indicate a significant difference from the control at P < 0.05 (according to Student’s t-test).

Fig. 4.

Different patterns of the PQ pool reduction in ics1, stn7, and WT Arabidopsis rosettes. (a) The redox status of the plastoquinone pool (expressed as 1–q _p) in different leaves of ics1, stn7, and the WT. Data represent an average from six different rosettes (n=6; grey shading indicates the 95% confidence intervals for the analysed genotypes). (b) Photos show false-colour images of 1–q _p for ics1, stn7, and the WT.

Fig. 5.

Microarray analysis of the ics1 mutant. (a) Volcano plot of microarray data. The log fold change is plotted on the x-axis (down-regulated genes on the left side, up-regulated on the right) and the negative log10 P-value is plotted on the y-axis. The black, solid line represents the P-value cut-off (0.05, Student’s t-test). Points above the line have P-values <0.05 and points below the line have P-values >0.05. The red points represent genes whose protein products are targeted to the chloroplasts. (b) Gene ontology (Cellular Component) of genes induced (red) and suppressed (green) in ics1. As a control, whole-genome categorization (grey) was used.