Sustained zeaxanthin-dependent thermal dissipation is induced by desiccation and low temperatures in the fern Polypodium virginianum.

Desiccation and low temperatures inhibit photosynthetic carbon reduction and, in combination with light, result in severe oxidative stress, thus, tolerant organisms must utilize enhanced photoprotective mechanisms to prevent damaging reactions from occurring. We sought to characterize the desiccation tolerance of the fern Polypodium virginianum and to assess the role of the xanthophyll cycle and sustained forms of thermal dissipation in its response to desiccation, as well as to low temperatures during winter. Our results demonstrate that P. virginianum is desiccation-tolerant and that it increases its utilization of sustained forms of zexanthin (Z)-dependent thermal dissipation in response to desiccation and low temperatures during winter. Experiments with detached fronds were conducted in dark and natural light conditions and demonstrated that some dark-formation of Z occurs in this species. In addition, desiccation in the light resulted in more pronounced declines in maximal photochemical efficiency (Fv /Fm ) and higher Z levels than desiccation in the dark, indicating a substantial fraction of the sustained reduction in Fv /Fm is due to Z-dependent sustained dissipation. Recovery from desiccation and from low temperatures exhibited biphasic kinetics with a more rapid phase (1-4 h), which was accompanied by an increase in minimal fluorescence yield (Fo ) but no change in Z, and a slower phase (up to 24 h) correlating with reconversion of Z to violaxanthin. These data suggest that two mechanisms of sustained thermal dissipation occur in response to desiccation and low temperatures, possibly corresponding to sustained forms of the energy-dependent and zeaxanthin-dependent mechanisms of dynamic thermal dissipation.

Is zeaxanthin needed for desiccation tolerance? Sustained forms of thermal dissipation in tolerant versus sensitive bryophytes.

Desiccation tolerant (DT) plants engage and disengage sustained forms of energy dissipation in response to desiccation and rehydration. This project sought to characterize the role of zeaxanthin and thylakoid protein phosphorylation status in sustained energy dissipation during desiccation in bryophytes with varying DT. Tolerant (Polytrichum piliferum, Dicranum species, Calliergon stramineum) and sensitive (Grimmia species, Schistidium rivulare, Sphagnum species) moss were desiccated in darkness or natural light conditions for up to three weeks. Desiccation caused pronounced reductions in Fv /Fm in all cases which was enhanced by light exposure during desiccation. Desiccation in darkness resulted in no accumulation of Z in any species, however, in natural light conditions there was significant accumulation of Z in tolerant but not sensitive species. Desiccation in natural light, relative to darkness, resulted in more pronounced reductions in Fo in tolerant but not sensitive species. Recovery of Fv /Fm upon rehydration occurred in two phases, a rapid phase (minutes) and a slower phase (hours). Increased time of desiccation, and light exposure, resulted in a reduction in the rapid phase. Desiccation in light conditions resulted in some accumulation of the phosphorylated form of the major light harvesting trimer (LHCII). Data are consistent with two mechanisms of sustained quenching, neither of which requires Z. However, when desiccation occurs in natural light conditions, accumulation of Z likely contributes to one or both of the sustained forms of dissipation. Increases in LHCII phosphorylation during desiccation are consistent with increased connectivity between the photosystems. The absence of Z formation in sensitive species may contribute to their lack of desiccation tolerance.

Could seasonally deteriorating environments favour the evolution of autogamous selfing and a drought escape physiology through indirect selection? A test of the time limitation hypothesis using artificial selection in Clarkia.

Background and Aims: The evolution of selfing from outcrossing may be the most common transition in plant reproductive systems and is associated with a variety of ecological circumstances and life history strategies. The most widely discussed explanation for these associations is the reproductive assurance hypothesis - the proposition that selfing is favoured because it increases female fitness when outcross pollen receipt is limited. Here an alternative explanation, the time limitation hypothesis, is addressed, one scenario of which proposes that selfing may evolve as a correlated response to selection for a faster life cycle in seasonally deteriorating environments. Methods: Artificial selection for faster maturation (early flowering) or for low herkogamy was performed on Clarkia unguiculata (Onagraceae), a largely outcrossing species whose closest relative, C. exilis, has evolved higher levels of autogamous selfing. Direct responses to selection and correlated evolutionary changes in these traits were measured under greenhouse conditions. Direct responses to selection on early flowering and correlated evolutionary changes in the node of the first flower, herkogamy, dichogamy, gas exchange rates and water use efficiency (WUE) were measured under field conditions. Key Results: Lines selected for early flowering and for low herkogamy showed consistent, statistically significant responses to direct selection. However, there was little or no evidence of correlated evolutionary changes in flowering date, floral traits, gas exchange rates or WUE. Conclusions: These results suggest that the maturation rate and mating system have evolved independently in Clarkia and that the time limitation hypothesis does not explain the repeated evolution of selfing in this genus, at least through its indirect selection scenario. They also suggest that the life history and physiological components of drought escape are not genetically correlated in Clarkia, and that differences in gas exchange physiology between C. unguiculata and C. exilis have evolved independently of differences in mating system and life history.

Characterization of light-dependent regulation of state transitions in gymnosperms.

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.

Seasonal changes in physiological performance in wild Clarkia xantiana populations: Implications for the evolution of a compressed life cycle and self-fertilization.

PREMISE OF THE STUDY: One explanation for the evolution of selfing, the drought escape hypothesis, proposes that self-fertilization may evolve under conditions of intensifying seasonal drought as part of a suite of traits that enable plants to accelerate the completion of their life cycle, thereby escaping late-season drought. Here, we test two fundamental assumptions of this hypothesis in Clarkia xantiana: (1) that a seasonal decline in precipitation causes an increase in drought stress and (2) that this results in changes in physiological performance, reflecting these deteriorating conditions. METHODS: We examined seasonal and interannual variation in abiotic environmental conditions (estimated by ambient temperature, relative humidity, predawn leaf water potentials, and carbon isotope ratios) and physiological traits (photosynthesis, conductance, transpiration, instantaneous water-use efficiency, ascorbate peroxidase and glutathione reductase activities, quantum yield of photosystem II, PSII potential efficiency) in field populations of C. xantiana in 2009 and 2010. KEY RESULTS: In both years, plants experienced intensifying drought across the growing season. Gas exchange rates decreased over the growing season and were lower in 2009 (a relatively dry year) than in 2010, suggesting that the temporal changes from early to late spring were directly linked to the deteriorating environmental conditions. CONCLUSIONS: Seasonal declines in transpiration rate may have increased survival by protecting plants from desiccation. Concomitant declines in photosynthetic rate likely reduced the availability of resources for seed production late in the season. Thus, the physiological patterns observed are consistent with the conditions required for the drought escape hypothesis.

Recovery kinetics of photochemical efficiency in winter stressed conifers: the effects of growth light environment, extent of the season and species.

Evergreens undergo reductions in maximal photochemical efficiency (F(v)/F(m)) during winter due to increases in sustained thermal energy dissipation. Upon removing winter stressed leaves to room temperature and low light, F(v)/F(m) recovers and can include both a rapid and a slow phase. The goal of this study was to determine whether the rapid component to recovery exists in winter stressed conifers at any point during the season in a seasonally extreme environment. Additional goals were to compare the effects of species, growth light environment and the extent of the winter season on recovery kinetics in conifers. Four species (sun and shade needle) were monitored during the winter of 2007/2008: eastern white pine (Pinus strobus), balsam fir (Abies balsamea), Taxus cuspidata and white spruce (Picea glauca). F(v)/F(m) was measured in the field, and then monitored indoors at room temperature and low light for 6 days. The results showed that all species showed a rapid component to recovery in early winter that disappeared later in the season in sun needles but was present in shade needles on most days monitored during winter. There were differences among species in the recovery kinetics across the season, with pine recovering the most slowly and spruce the most quickly. The results suggest an important role for the rapidly reversible form of energy dissipation in early winter, as well as important differences between species in their rate of recovery in late winter/early spring which may have implications for spring onset of photosynthesis.

Effects of lincomycin on PSII efficiency, non-photochemical quenching, D1 protein and xanthophyll cycle during photoinhibition and recovery.

Leaves of Parthenocissus quinquefolia (L.) Planch. (Virginia creeper) were treated with lincomycin (an inhibitor of chloroplast-encoded protein synthesis), subjected to a high-light treatment and allowed to recover in low light. While lincomycin-treated leaves had similar characteristics as controls after a 1 h exposure to high light, total D1 levels in lincomycin-treated leaves were half those in controls at the end of the recovery period. In addition, lincomycin delayed recovery of maximal PSII efficiency of open centers (ratio of variable to maximal chlorophyll fluorescence, F v / F m) and of estimated PSII photochemistry rate upon return to low light subsequent to the high-light treatment. Furthermore, lincomycin treatment slowed the removal of zeaxanthin (Z) and antheraxanthin (A) during recovery in low light, and the level of thermal energy dissipation (non-photochemical fluorescence quenching, NPQ) remained elevated. In lincomycin-treated leaves infiltrated with the uncoupler nigericin immediately after high-light exposure, thermal energy dissipation, sustained with lincomycin alone, declined quickly to control levels. In summary, lincomycin treatment affected not only D1 protein turnover but also xanthophyll-cycle operation and thermal-energy dissipation. The latter effect was apparently a result of the maintenance of a high trans-thylakoid proton gradient. Similar effects were also seen subsequent to short-term exposures to high light in lincomycin-treated Spinacia oleracea L. (spinach) leaves. In contrast, lincomycin treatments under low-light levels did not induce Z formation or NPQ. These results suggest that lincomycin has the potential to lower PSII efficiency (F v / F m) through inhibition of NPQ relaxation and Z + A removal subsequent to high-light exposures.

Overexpression of violaxanthin de-epoxidase: properties of C-terminal deletions on activity and pH-dependent lipid binding.

Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A full-length Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the specific activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity. The difficulties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.