Marianna Krol

Photosynthesis, photoinhibition and low temperature acclimation in cold tolerant plants.

Cold acclimation requires adjustment to a combination of light and low temperature, conditions which are potentially photoinhibitory. The photosynthetic response of plants to low temperature is dependent upon time of exposure and the developmental history of the leaves. Exposure of fully expanded leaves of winter cereals to short-term, low temperature shiftsinhibits whereas low temperature growthstimulates electron transport capacity and carbon assimilation. However, the photosynthetic response to low temperature is clearly species and cultivar dependent. Winter annuals and algae which actively grow and develop at low temperature and moderate irradiance acquire a resistance to irradiance 5- to 6-fold higher than their growth irradiance. Resistance to short-term photoinhibition (hours) in winter cereals is a reflection of the increased capacity to keep QA oxidized under high light conditions and low temperature. This is due to an increased capacity for photosynthesis. These characteristics reflect photosynthetic acclimation to low growth temperature and can be used to predict the freezing tolerance of cereals. It is proposed that the enhanced photosynthetic capacity reflects an increased flux of fixed carbon through to sucrose in source tissue as a consequence of the combined effects of increased storage of carbohydrate as fructans in the vacuole of leaf mesophyll cells and an enhanced export to the crown due to its increased sink activity. Long-term exposure (months) of cereals to low temperature photoinhibition indicates that this reduction of photochemical efficiency of PS II represents a stable, long-term down regulation of PS II to match the energy requirements for CO2 fixation. Thus, photoinhibition in vivo should be viewed as the capacity of plants to adjust photosynthetically to the prevailing environmental conditions rather than a process which necessarily results in damage or injury to plants. Not all cold tolerant, herbaceous annuals use the same mechanism to acquire resistance to photoinhibition. In contrast to annuals and algae, overwintering evergreens become dormant during the cold hardening period and generally remain susceptible to photoinhibition. It is concluded that the photosynthetic response to low temperatures and susceptibility to photoinhibition are consequences of the overwintering strategy of the plant species.

Photosynthetic Redox Imbalance Governs Leaf Sectoring in the Arabidopsis thaliana Variegation Mutants immutans, spotty, var1, and var2

We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R2 = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype.

IMMUTANS Does Not Act as a Stress-Induced Safety Valve in the Protection of the Photosynthetic Apparatus of Arabidopsis during Steady-State Photosynthesis

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O2 to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P700 to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6×) and 16 (OE-16×) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1). Similar results were observed either after 3-d cold stress at 5°C or after full-leaf expansion at 5°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1. Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25°C or 5°C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P700+ during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.

Abscisic acid induced protection against photoinhibition of PSII correlates with enhanced activity of the xanthophyll cycle

The exogenous application of abscisic acid (ABA) to barley seedlings resulted in partial protection of the PSII photochemistry against photoinhibition at low temperature, the effect being most pronounced at 10−5 M ABA. This was accompanied by higher photochemical quenching (qP) in ABA‐treated leaves. A considerable increase (122%) in the amount of total carotenoids and xanthophylls (antheraxanthin, violaxanthin and zeaxanthin) was also found in the seedlings subjected to ABA. The activity of the xanthophyll cycle measured by the epoxidation state of xanthophylls under high‐light treatment was higher in ABA‐treated plants compared with the control. This corresponds to a higher value (0.411) of non‐photochemical quenching (qNP) observed in ABA‐treated than in control (0.306) leaves.

Effect of long-term photoinhibition on growth and photosynthesis of cold-hardened spring and winter wheat

The effect of repeated exposure to high light (1200 μmol · m−2 · s−1 photosynthetic photon flux density, PPFD) at 5° C was examined in attached leaves of cold-grown spring (cv. Katepwa) and winter (cv. Kharkov) wheat (Triticum aestivum L.) over an eight-week period. Under these conditions, Kharkov winter wheat exhibited a daily reduction of 24% in FV/FM (the ratio of variable to maximal fluorescence in the dark-adapted state), in contrast to 41% for cold-grown Katepwa spring wheat. Both cultivars were able to recover from this daily suppression of FV/FM such that the leaves exhibited an average morning FV/FM of 0.651 ± 0.004. Fluorescence measurements made under steady-state conditions as a function of irradiance from 60 to 2000 μmol · m−2 · s−1 indicated that the yield of photosystem II (PSII) electron transport under light-saturating conditions was the same for photoinhibited and control cold-grown plants, regardless of cultivar. Repeated daily exposure to high light at low temperature did not increase resistance to short-term photoinhibition, although zeaxanthin levels increased by three- to fourfold. In addition, both cultivars increased the rate of dry-matter accumulation, relative to control plants maintained at 5° C and 250 μmol · m−2 · s−1 PPFD (10% and 28% for Katepwa and Kharkov, respectively), despite exhibiting suppressed fv/fm and reduced photon yields for O2 evolution following daily high-light treatments. Thus, although photosynthetic efficiency is suppressed by a longterm, photoinhibitory treatment, light-saturated rates of photosynthesis are sufficiently high during the high-light treatment to offset any reduction in photochemical efficiency of PSII. We suggest that in these cold-tolerant plants, photoinhibition of PSII may represent a longterm, stable, down-regulation of photochemistry to match the overall photosynthetic demand for ATP and reducing equivalents.

Chloroplast redox imbalance governs phenotypic plasticity: the “grand design of photosynthesis” revisited

Sunlight, the ultimate energy source for life on our planet, enters the biosphere as a direct consequence of the evolution of photoautotrophy. Photoautotrophs must balance the light energy absorbed and trapped through extremely fast, temperature-insensitive photochemistry with energy consumed through much slower, temperature-dependent biochemistry and metabolism. The attainment of such a balance in cellular energy flow between chloroplasts, mitochondria and the cytosol is called photostasis. Photoautotrophs sense cellular energy imbalances through modulation of excitation pressure which is a measure of the relative redox state of QA, the first stable quinone electron acceptor of photosystem II reaction centers. High excitation pressure constitutes a potential stress condition that can be caused either by exposure to an irradiance that exceeds the capacity of C, N, and S assimilation to utilize the electrons generated from the absorbed energy or by low temperature or any stress that decreases the capacity of the metabolic pathways downstream of photochemistry to utilize photosynthetically generated reductants. The similarities and differences in the phenotypic responses between cyanobacteria, green algae, crop plants, and variegation mutants of Arabidopsis thaliana as a function of cold acclimation and photoacclimation are reconciled in terms of differential responses to excitation pressure and the predisposition of photoautotrophs to maintain photostasis. The various acclimation strategies associated with green algae and cyanobacteria versus winter cereals and A. thaliana are discussed in terms of retrograde regulation and the “grand design of photosynthesis” originally proposed by Arnon (1982).

Iron Deficiency in Cyanobacteria Causes Monomerization of Photosystem I Trimers and Reduces the Capacity for State Transitions and the Effective Absorption Cross Section of Photosystem I in Vivo

The induction of the isiA (CP43′) protein in iron-stressed cyanobacteria is accompanied by the formation of a ring of 18 CP43′ proteins around the photosystem I (PSI) trimer and is thought to increase the absorption cross section of PSI within the CP43′-PSI supercomplex. In contrast to these in vitro studies, our in vivo measurements failed to demonstrate any increase of the PSI absorption cross section in two strains (Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803) of iron-stressed cells. We report that iron-stressed cells exhibited a reduced capacity for state transitions and limited dark reduction of the plastoquinone pool, which accounts for the increase in PSII-related 685 nm chlorophyll fluorescence under iron deficiency. This was accompanied by lower abundance of the NADP-dehydrogenase complex and the PSI-associated subunit PsaL, as well as a reduced amount of phosphatidylglycerol. Nondenaturating polyacrylamide gel electrophoresis separation of the chlorophyll-protein complexes indicated that the monomeric form of PSI is favored over the trimeric form of PSI under iron stress. Thus, we demonstrate that the induction of CP43′ does not increase the PSI functional absorption cross section of whole cells in vivo, but rather, induces monomerization of PSI trimers and reduces the capacity for state transitions. We discuss the role of CP43′ as an effective energy quencher to photoprotect PSII and PSI under unfavorable environmental conditions in cyanobacteria in vivo.

Acclimation to temperature and irradiance modulates PSII charge recombination.

Acclimation of wild type and the chlorina F2 mutant of barley to either high light or low temperature results in a 2‐ to 3‐fold increase in non‐photochemical quenching which occurred independently of either energy‐dependent quenching (qE), xanthophyll cycle‐mediated antenna quenching or state transitions. Results of in vivo thermoluminescence measurements used to address this conundrum indicated that excitation pressure regulates the temperature gap for S 2 Q B ‐ and S 2 Q A ‐ charge recombinations within photosystem II reaction centers. This is discussed in terms of photoprotection through non‐radiative charge recombination.

Adjustment of thylakoid plastoquinone content and Photosystem I electron donor pool size in response to growth temperature and growth irradiance in winter rye (Secale cereale L.)

Winter rye (Secale cereale L. cv Musketeer) grown at 5 °C/250 µmol photons m−2 s−1 exhibited a relative reduction state of PS II comparable to that of rye grown at 20 °C but high light (800 µmol photons m−2 s−1) (1-qP = 0.32) whereas winter rye grown at 20 °C/250 µmol photons m−2 s−1 exhibited values of 1-qP (≈ 0.15) comparable to plants grown at 5 °C but low light (50 µmol photons m−2 s−1). The apparent size of the electron donor pool to PS I, estimated either in vivo or in vitro in the presence of methylviologen by ΔA820 was positively correlated with the relative reduction state of PS II under the steady-state growth conditions. Immunoblotting of rye thylakoid polypeptides indicated that the relative contents of Lhcb1, Lhcb2, D1, Cyt f, PC, PsaA/PsaB heterodimer and the β-subunit of ATPase complex exhibited minimal changes on a Chl basis. In contrast, a 2-fold increase in plastoquinone A content was associated with increasing growth irradiance at growth temperatures of either 5 or 20 °C. We suggest that the increases in the apparent size of the electron donor pool to PS I associated with rye grown at either 5 °C/250 µmol photons m−2 s−1or 20 °C/800 µmol photons m−2 s−1 may be explained by an increased thylakoid plastoquinone A content, coupled with possible enhanced PS I cyclic electron transport and/or increased capacity for electron donation from the stroma to the intersystem electron transport chain. The results are discussed with respect to photosynthetic adjustment to changes in PS II ‘excitation pressure’ in winter rye.

The Antarctic psychrophile, Chlamydomonas raudensis Ettl (UWO241) (Chlorophyceae, Chlorophyta), exhibits a limited capacity to photoacclimate to red light

The psychrophilic Antarctic alga, Chlamydomonas raudensis Ettl (UWO241), grows under an extreme environment of low temperature and low irradiance of a limited spectral quality (blue‐green). We investigated the ability of C. raudensis to acclimate to long‐term imbalances in excitation caused by light quality through adjustments in photosystem stoichiometry. Log‐phase cultures of C. raudensis and C. reinhardtii grown under white light were shifted to either blue or red light for 12 h. Previously, we reported that C. raudensis lacks the ability to redistribute light energy via the short‐term mechanism of state transitions. However, similar to the model of mesophilic alga, C. reinhardtii, the psychrophile retained the capacity for long‐term adjustment in energy distribution between PSI and PSII by modulating the levels of PSI reaction center polypeptides, PsaA/PsaB, with minimal changes in the content of the PSII polypeptide, D1, in response to changes in light quality. The functional consequences of the modulation in PSI/PSII stoichiometry in the psychrophile were distinct from those observed in C. reinhardtii. Exposure of C. raudensis to red light caused 1) an inhibition of growth and photosynthetic rates, 2) an increased reduction state of the intersystem plastoquinone pool with concomitant increases in nonphotochemical quenching, 3) an uncoupling of the major light‐harvesting complex from the PSII core, and 4) differential thylakoid protein phosphorylation profiles compared with C. reinhardtii. We conclude that the characteristic low levels of PSI relative to PSII set the limit in the capacity of C. raudensis to photoacclimate to an environment enriched in red light.