Fig 1.
Effect of Sclerotinia infection on NPQ.
(A) Chlorophyll fluorescence imaging showing changes in Fv/Fm and NPQ in Sclerotinia-infected Arabidopsis leaves. After inoculation with Sclerotinia, plants were dark-adapted for 1 h prior to measurement of chlorophyll fluorescence. Because NPQ values always higher than one but rarely exceeded four, NPQ/4 was displayed by pseudo-color with values ranging from zero (black) to one (purple). (B, C) Examples for statistical analysis of NPQ in the entire inoculated region (B) and the leading edge (C). (D, E) Quantitative analysis of the values of NPQ in Sclerotinia-infected entire region (D) and leading edge (E). Values are means ± SE of three replicates. (F) Infection phenotypes that related NPQ changes after inoculated with Sclerotinia. Inset: the corresponding NPQ/4 image. Arrows indicate the water soaking phenotypes. Scale bars in original (upper) and magnified (below) pictures are 2 mm and 0.5 mm, respectively. (G) Scanning electron micrographs showing detailed changes on the leaf surface after inoculated with Sclerotinia. Pictures were obtained with a cold field scanning electron microscope at an accelerating voltage of 3.0 kV. Red arrows indicate the slight broken cuticle; Blue arrows indicate mycelial cells; Purple arrows indicate amorphous structures that might be derived from PDA plugs. Scale bars in original and magnified images are 50 μm and 5 μm, respectively. (H) A model to explain NPQ variation at different infection stages and regions. The growing hyphae are painted with yellow color. Drawing of plant cells with blue lines represent un-infected zone; red lines represent leading edge of infected zone; dark lines meant severely damaged tissue.
Fig 2.
Dynamics of NPQ in Sclerotinia-infected leaves.
(A-C) Chlorophyll fluorescence imaging showing changes of NPQ in Sclerotinia-inoculated leaves under (A) different intensity of actinic light, (B) high light (725 μmol photons m-2 s-1), and (C) low light (133 μmol photons m-2 s-1). PAR, photosynthetically active radiation (μmol photons m-2 s-1). Light was switched off at 4 min (black arrow). False colour code is depicted at the right of the image. (D) Kinetics of NPQ in Sclerotinia-infected leaves under different light intensity. (E, F) Induction and relaxation kinetics of NPQ in Sclerotinia-infected zone under high light (E) and low light (F). Each curve represents the average of three replicates ± SE.
Fig 3.
Role of Sclerotinia-secreted oxalate in the NPQ increase.
(A) Scanning electron micrographs showing calcium oxalate crystal formed at the Sclerotinia-inoculated site. Red arrows indicate calcium oxalate crystal. (B, C) Confocal images showing positional pH changes in tissues infected with wild-type Sclerotinia or the oxalate-deficient A2 mutant. Tissue acidification was determined by lysosensor green DND-189 (B) and acridine orange (C), respectively. The fluorescence ratio of red to green emissions of acridine orange was displayed with pseudo-color. Bars = 50 μm. (D) False colour images showing Fv/Fm and NPQ/4 after challenge with wild-type Sclerotinia (Circle 1) and A2 mutant (Circle 2). (E) Dynamics of NPQ in A2 mutant and wild-type (WT) Sclerotinia-infected regions. Values represent average of three replicates ± SE.
Fig 4.
Effect of Sclerotinia invasion on the xanthophyll cycle.
(A, B) Impact of mutations in PsbS (A) or vde (B) on the dynamics of NPQ induced by Sclerotinia. Kinetics of NPQ were recorded in Sclerotinia-infected npq4-1 (PsbS) and npq1-2(vde) mutants, respectively. (C) The inhibitory effects of DTT on the kinetics of NPQ induced by Sclerotinia. NPQ was measured in dark-adapted Col-0 leaves after vacuum pre-infiltration with DTT (10 μM). (D) The component of zeaxanthin-related NPQ at Sclerotinia-infected regions. The amplitude of zeaxanthin-related NPQ was expressed as total NPQ minus NPQ+DTT. Data shown are the average of three replicates ± SE.
Table 1.
Pigment composition of Sclerotinia-infected plants.
Fig 5.
Impact of Sclerotinia invasion on ABA biosynthesis.
(A) Effect of Sclerotinia invasion on the expression of ABA biosynthesis genes. The expression of target genes was determined by qPCR. (B) Immuno-cytochemical localization of ABA in longitudinal sections of Arabidopsis leaves. Bars = 0.2 mm. (C) Measurement of ABA levels in Sclerotinia-inoculated leaf regions with an ELISA Kit. (D) A2 mutant invasion caused leaf yellowing surrounding the necrotic lesions. Images were captured at 24 h after inoculation with A2 mutant. (E) Quantitative analysis of the content of ABA in the yellowing regions. (F) Mutation of VDE alleviated the decrease in ABA levels in Sclerotinia-infected npq1-2 leaves. WT, wild-type Sclerotinia; A2, oxalate-deficient A2 mutant. (**), Student’s t-test significant at P< 0.01; (*), P< 0.05. Different letters indicate statistically significant differences (Duncan’s multiple range tests; P< 0.05). Data are average of three replicates± SE.
Fig 6.
Efficiency of ABA in inducing plant resistance against Sclerotinia.
(A, B) Effect of ABA on disease symptoms (A) and lesion areas (B) in Col-0 and npq1-2 plants after inoculation with wild-type Sclerotinia. Leaves were incubated with Sclerotinia for 24 h in the presence or absence of 100 μM ABA. (C) Defects in ABA signaling increased susceptibility of npq1-2/abi4-1 and npq1-2/aba2-3 relative to npp1-2 upon wild-type Sclerotinia infection. Lesion areas were measured at 24 h after inoculation with wild-type Sclerotinia. (D) Defects in ABA signaling increased susceptibility of npq1-2/abi4-1 and npq1-2/aba2-3 plants to oxalate-deficient A2 mutant. Lesion areas caused by the A2 mutant were measured at 48 h. Values represent means ± SE of at least 10 lesions. Different letters indicate statistically significant differences (Duncan’s multiple range tests; P < 0.05). (E, F) Pretreatment with ABA induced O2- formation (E) and callose deposition (F) at the advancing edge of necrotic areas. O2- was stained with NBT, forming blue formazan. Red arrows indicate callose deposition. Scale bar = 2 mm. Images shown are representative.
Fig 7.
Proposed model for the interplay of the xanthophyll cycle and plant resistance to Sclerotinia.
Sclerotinia secretes oxalate to acidify the infected tissues, which results in increased lumen acidification. Subsequently, the low lumen pH activates NPQ by protonating PsbS protein and VDE. The latter convert violaxanthin (V) to zeaxanthin (Z) via the intermediate antheraxanthin (A) in the xanthophyll cycle. Elevated NPQ and zeaxanthin accumulation have the potential to affect chloroplast redox singling pathways to lower oxidation-derived oxylipins (like jasmonic acid, JA) formation [10–11,77]. On the other hand, the decrease in precursor violaxanthin limits ABA biosynthesis. These aspects, in turn, affects tissue defense responses like ROS induction and callose deposition, which increases plant susceptibility to Sclerotinia.