Fig 1.
Ethylene regulates induction of NPQ.
eto1-1, ctr1-3, ein2-5, and WT Arabidopsis were grown under 250 PFD for 3.5 weeks. The induction of NPQ in dark-adapted plants exposed to 336 PFD was measured at (A) 20 sec, (B) 60 sec, (C) 2 min, (D) 10 min, (E) 15 min using chlorophyll fluorescence video imaging. (F) Inhibition of PSII quantum yield at 20 min of recovery in the dark following a 25 min light exposure to 336 PFD was measured as 1—(sample Fv/Fm/dark-adapted Fv/Fm). The level of NPQ induced is presented as false color images according to the color bar below the images. (G) NPQ and Fv/Fm were measured in leaves of 3.5 week-old WT, eto1-1, and ctr1-3 plants grown at 250 PFD following exposure to 400 PFD. Fast and slow relaxation of NPQ (i.e., NPQf and NPQs, respectively) were measured following exposure to 1800 PFD for 30 min. The data reported are the average of five biological replicates.
Fig 2.
Violaxanthin de-epoxidation is reduced in eto1-1 and ctr1-3 under saturating light conditions.
(A) Xanthophyll pigments were isolated from leaves of plants (grown at 250 PFD) dark-adapted for 16 hr, or treated with 500 PFD for the times indicated. The pigments were quantitated by HPLC and normalized to chlorophyll a (i.e., μg/mg Chl a). The de-epoxidation status, i.e., (0.5A + Z)/(V + A + Z) was determined from the amounts of violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) and is included below each pie chart. (B) Graphical display of the de-epoxidation state of the xanthophyll pigments from (A). The data reported are the average of three biological replicates.
Fig 3.
Increased ethylene signaling represses VDE and PsbS expression.
(A) The xanthophyll cycle. NPQ1 encodes violaxanthin de-epoxidase (VDE) whereas NPQ2 encodes zeaxanthin epoxidase (ZE). (B) The pool sizes for Asc, DHA, total ascorbate (i.e., Asc + DHA), and the Asc redox state were measured in leaves of 4 week-old eto1-1, ctr1-3, and WT plants. (C) qPCR analysis of NPQ1 mRNA in leaves of 3 week-old plants. (D) qPCR analysis of PsbS mRNA in leaves of 3 week-old plants. (E) VDE and PsbS protein levels were measured by Western analysis in leaves of 3 week-old plants. Western analysis of the large subunit of Rubisco served as a control. Loading was on an equal chlorophyll basis. (F) VDE and ZE enzyme activity were measured in leaves of 3 week-old eto1-1, ctr1-3, and WT plants. The data reported are the average and standard deviation of three biological replicates.
Fig 4.
Ethylene represses NPQ1 promoter activity.
(A) Ethylene evolution from WT Arabidopsis and transgenic seedlings containing NPQ1::Luc grown for 2 weeks in the absence or presence of 20 μM ACC. (B) Luciferase expression from transgenic Arabidopsis seedlings containing the NPQ1::Luc transgene grown at 100 PFD for 2 weeks in the presence of AgNO3 (white bars) or 20 μM ACC (black bars). Transgenic Arabidopsis containing a 35S::Luc construct was included as a control under the same growth conditions. The same lines grown at 100 PFD were also transferred to 250 PFD for 24 hr prior to assaying. Bars with asterisks indicate significant difference (p < 0.01) relative to the same line grown in the presence of AgNO3. (C) Luciferase expression from of 3 week-old transgenic WT, eto1-1, ctr1-3, and ein2-5 seedlings containing the NPQ1::Luc or 35S::Luc transgenes grown at 250 PFD.
Fig 5.
The defect in CO2 assimilation in eto1-1 is corrected by increasing CO2 availability.
(A) The rate of CO2 assimilation was measured in light-adapted WT, ein2-5, and eto1-1 plants at 400 PFD as a function of CO2 concentration. The rate of CO2 assimilation is plotted against the internal CO2 concentration (Ci). WT (filled diamonds); ein2-5 (filled triangles); eto1-1 (filled circles). (B) Induction of NPQ was measured in dark-adapted WT and eto1-1 plants grown at 250 PFD and exposed to 400 PFD under ambient or 1800 ppm CO2. WT (filled diamonds); eto1-1 (filled circles); WT (open diamonds); and eto1-1 (open circles).
Table 1.
Carbonic anhydrase activity in ethylene mutants.
Table 2.
The transthylakoid membrane pH gradient is reduced in ethylene mutants.
Fig 6.
Restoring VDE expression corrects the aberrant NPQ induction and violaxanthin de-epoxidation in eto1-1 and ctr1-3 plants.
(A) The induction of NPQ in dark-adapted WT, eto1-1, and ctr1-3 plants with or without the 35S::NPQ1 transgene following their exposure to 396 PFD for 60 sec using chlorophyll fluorescence video imaging. The level of NPQ is presented as false color images as indicated by the color bar below the image. Dark-adapted eto1-1, ctr1-3, and WT (grown at 250 PFD) without (B) or with (C) the 35S::NPQ1 transgene were treated with 1000 PFD for the times indicated. Xanthophyll pigments were quantitated by HPLC and normalized to chlorophyll a (i.e., μg/mg Chl a). (D) The kinetics of the rate of de-epoxidation is shown.
Table 3.
Restoring VDE expression to eto1-1 and ctr1-3 reduces the rate of O2.- production.
Table 4.
Restoring VDE expression to eto1-1 and ctr1-3 increases the transthylakoid membrane pH gradient.
Table 5.
Increased ethylene signaling increases the electron requirement per molecule CO2 fixed.
Fig 7.
Restoring VDE expression or elevated CO2 reverses the qI component of NPQ in eto1-1 and ctr1-3.
The aberrant accumulation of NPQ in eto1-1 leaves is corrected by increasing CO2. Induction of NPQ was measured in dark-adapted WT, eto1-1, and ctr1-3 plants grown at 250 PFD and exposed to 400 PFD under (A) ambient or (B) elevated CO2 (i.e., 1800 ppm CO2). WT (open diamonds); WT T:NPQ1 (filled diamonds); eto1-1 (open circles); eto1-1 T:NPQ1 (filled circles); and ctr1-3 (open triangles); and ctr1-3 T:NPQ1 (filled triangles). The data reported are the average of four replicates.
Fig 8.
Restoring VDE expression reverses the aberrant relaxation of NPQ in eto1-1 and ctr1-3 following exposure to high-light stress.
(A) Fast and slow relaxation of NPQ (i.e., NPQf and NPQs, respectively) were measured in adult leaves of eto1-1 and ctr1-3 plants, without or with the 35S::NPQ1 transgene, following exposure to 1800 PFD for 30 min. WT plants without or with the 35S::NPQ1 transgene were included in each analysis. Measurements were made prior to the appearance of the inflorescence. Values for each are presented below each pie chart as is the dark-adapted Fv/Fm. Values were determined from four replicates. The recovery of (B) eto1-1 and eto1-1 T:NPQ1 plants or (C) ctr1-3 and ctr1-3 T:NPQ1 plants from a 2 hr exposure to sunlight (i.e., 1900 PFD) was determined by measuring Fv/Fm over time following the transfer of plants to darkness to facilitate recovery. The data was expressed relative to dark-adapted Fv/Fm value in order to make direct comparisons between lines. WT (filled diamonds); WT T:NPQ1 (open diamonds); eto1-1 and ctr1-3 (open squares); eto1-1 T:NPQ1 and ctr1-3 T:NPQ1 (filled squares). The data reported are the average of six replicates.
Table 6.
Restoring VDE expression in eto1-1 and ctr1-3 does not alter α-tocopherol content.
Fig 9.
Restoring VDE expression reverses the small growth phenotype of eto1-1 plants.
(A) eto1-1 and WT plants without or with the 35S::NPQ1 transgene were grown at 1200 PFD for 3.5 weeks. (B) Comparison of adult rosette leaves of eto1-1 and WT plants with or without the 35S::NPQ1 transgene grown at 1200 PFD. (C) Fresh and dry weight of eto1-1 and WT plants without or with the 35S::NPQ1 transgene grown at 1200 PFD. (D) eto1-1 and WT plants without or with the 35S::NPQ1 transgene were grown under 50 PFD for 3.5 weeks. (E) Comparison of adult rosette leaves of eto1-1 and WT plants with or without the 35S::NPQ1 transgene grown under 50 PFD. (F) Fresh and dry weight of eto1-1 and WT plants without or with the 35S::NPQ1 transgene grown at 50 PFD. (G) Every leaf from eto1-1 and WT plants without or with the 35S::NPQ1 transgene grown for 3.5 weeks at 1200 PFD.
Table 7.
Increasing VDE expression does not affect ethylene production or responsiveness.