Characterization of light-dependent regulation of state transitions in gymnosperms

Abstract

The goal of this study was to characterize the light-dependent regulation of state transitions in gymnosperms. Two species of conifer were examined: eastern white pine (Pinus strobus L.) and white spruce [Picea glauca (Moench) Voss], as well as the angiosperm pumpkin (Cucurbita pepo L. subsp. pepo). Both diurnal time courses in the field and manipulated light experiments in growth chambers were conducted. Results from chlorophyll fluorescence analysis indicated that pumpkin was able to use a larger fraction of absorbed light to drive photochemistry and retain a lower reduction state at a given light intensity relative to the conifers. Results from western blots using anti-phosphothreonine demonstrate that in field conditions, conifers maintained higher light-harvesting complex II (LHCII) phosphorylation than pumpkin; however, this was likely due to a more variable light environment. Manipulated light experiments showed that general patterns of light-dependent LHCII phosphorylation were similar in conifers and pumpkin, with low levels of LHCII phosphorylation occurring in darkness and maximal levels occurring in low light conditions. However, high light-dependent dephosphorylation of LHCIII appears to be regulated differently in conifers, with conifers maintaining phosphorylation of LHCII proteins at higher excitation pressure compared with pumpkin. Additionally, spruce needles maintained relatively high phosphorylation of LHCII even in very high light conditions. Our results suggest that this difference in dephosphorylation of LHCII may be due to differences in the stromal redox status in spruce relative to pine and pumpkin.

Keywords: conifer; light regulation; thylakoid protein phosphorylation.

Figure 1.

Light intensity (photosynthetic photon flux density, PPFD) at leaf level at the time of sampling (a) and ΦPSII or F_v/F_m (b) obtained over the course of a portion of 2 days for pumpkin (open circles), pine (filled squares) and spruce (open triangles). Time points for samples taken predawn until 11:00 h were collected in 13 July 2010, and the time points from 13:30 h until the end of the day were collected on 15 July 2010. Data represent averages ± SD (n = 3).

Figure 2.

Example blots showing the phosphorylation status of thylakoid proteins of PSII (CP43, D2 and D1) as well as phospho-LHCII in samples collected from pumpkin, pine and spruce over the course of the day.

Figure 3.

The relative phosphorylation of individual thylakoid proteins from samples collected under different light intensities for pumpkin (white bars), pine (gray bars) and spruce (black bars). Samples were grouped into categories of light intensity [dark, low light, medium light and high light (0, 20–100, 150–700 and >1000 µmol photons m⁻² s⁻¹)] in order to compare general light intensity effects. For these data, the time point showing the highest phosphorylation of LHC was normalized to 1, and all other time periods were expressed as a fraction of that value. The number of time points pooled and the respective light intensities during collection were as follows: 20–100 µmol photons m⁻² s⁻¹ range: pumpkin (3) 27, 63, 85, pine (2) 20, 60, spruce (3) 30, 50, 75; 150–700 µmol photons m⁻² s⁻¹ range: pumpkin (1) 660, pine (2) 250, 450, spruce (2) 175, 400; >1000 µmol photons m⁻² s⁻¹ range: pumpkin (3) 1100, 1380, 1465, pine (3) 1000, 1150, 1300, spruce (2) 1225, 1812. Where two or more samples were pooled, data represent averages ± SD.

Figure 4.

The chlorophyll fluorescence parameters ΦPSII or F_v/F_m (a), 1 − q_L (b) and the relative phosphorylation of LHCII (c) for samples from pumpkin (open circles), pine (filled squares) and spruce (open triangles) as a function of light intensity. Data are averages ± SD of three experiments.

Figure 5.

The activation state of the enzyme NADP-MDH, which is an indirect measure of the redox status of the chloroplast stroma, as a function of light intensity. Values closer to 1 indicate a higher stromal reduction state. Data are averages ± SD of three experiments for pumpkin (open circles), pine (filled squares) and spruce (open triangles).

Figure 6.

1 − q_L (a) or NADPH-MDH activation state (b) as a function of relative LHCII phosphorylation levels in pumpkin (open circles), pine (filled squares) and spruce (open triangles). The lines depict estimates of best fit for a linear regression in (a) and the curves depict estimates of best fit for a second-order polynomial in (b) using Graphpad Prism.