Photosystem II energy use,non-photochemical quenching and the xanthophyll cycle in Sorghumbicolor grown under drought and free-air CO2 enrichment(FACE) conditions

The present study was carried out to test the hypothesis thatelevated atmospheric CO₂ (Ca) will alleviate over‐excitationof the C₄ photosynthetic apparatus and decrease non‐photochemicalquenching (NPQ) during periods of limited water availability. Chlorophyll a fluorescencewas monitored in Sorghum bicolor plants grown under a free‐aircarbon‐dioxide enrichment (FACE) by water‐stress (Dry) experiment.Under Dry conditions elevated Ca increased the quantum yield ofphotosystem II (φPSII) throughout the day throughincreases in both photochemical quenching coefficient (q_p)and the efficiency with which absorbed quanta are transferred toopen PSII reaction centres (F_v′/F_m′).However, in the well‐watered plants (Wets) FACE enhanced φPSIIonly at midday and was entirely attributed to changes in F_v′/F_m′_. Underfield conditions, decreases in φPSII under Dry treatmentsand ambient Ca corresponded to increases in NPQ but the de‐epoxidation stateof the xanthophyll pool (DPS) showed no effects. Water‐stress didnot lead to long‐term damage to the photosynthetic apparatus asindicated by φPSII and carbon assimilation measuredafter removal of stress conditions. We conclude that elevated Caenhances photochemical light energy usage in C₄ photosynthesisduring drought and/or midday conditions. Additionally,NPQ protects against photo‐inhibition and photodamage. However,NPQ and the xanthophyll cycle were affected differently by elevatedCa and water‐stress.     

Arabidopsis plants lacking PsbS protein possess photoprotective energy dissipation

It is commonly accepted that the photosystem II subunit S protein, PsbS, is required for the dissipation of excess light energy in a process termed ‘non‐photochemical quenching’ (NPQ). This process prevents photo‐oxidative damage of photosystem II (PSII) thus avoiding photoinhibition which can decrease plant fitness and productivity. In this study Arabidopsis plants lacking PsbS (the npq4 mutant) were found to possess a competent mechanism of excess energy dissipation that protects against photoinhibitory damage. The process works on a slower timescale, taking about 1 h to reach the same level of NPQ achieved in the wild type in just a few minutes. The NPQ in npq4 was found to display very similar characteristics to the fast NPQ in the wild type. Firstly, it prevented the irreversible light‐induced closure of PSII reaction centres. Secondly, it was uncoupler‐sensitive, and thus triggered by the ΔpH across the thylakoid membrane. Thirdly, it was accompanied by significant quenching of the fluorescence under conditions when all PSII reaction centres were open (F_o state)_. Fourthly, it was accompanied by NPQ‐related absorption changes (ΔA535). Finally, it was modulated by the presence of the xanthophyll cycle carotenoid zeaxanthin. The existence of a mechanism of photoprotective energy dissipation in plants lacking PsbS suggests that this protein plays the role of a kinetic modulator of the energy dissipation process in the PSII light‐harvesting antenna, allowing plants to rapidly track fluctuations of light intensity in the environment, and is not the primary cause of NPQ or a direct carrier of the pigment acting as the non‐photochemical quencher.     

依赖于叶黄素循环的热耗散对茶树叶片防御强光破坏的相关试验研究

经叶黄素循环抑制剂——二硫苏糖醇(DIT)处理的茶树叶片,以850μmol．m^-2．s^-1的PFD照射120min后,福鼎大白茶的叶黄素循环组分中的环氧玉米黄素(A)和玉米黄素(Z)含量之和降低了76．5％,结果导致非光化学猝灭(NPQ)、光系统Ⅱ(PSⅡ)的光化学效率(Fv／Fm)、光化学猝灭系数(qP)、PSⅡ实际光化学量子效率(ψPSⅡR)和光合电子传递速率(ETR)明显下降,而F0显著上升,暗恢复后Fv／Fm恢复程度小于未经DIT处理的叶片。自然光强下,NPQ与与叶黄素循环的脱环氧化程度(A Z)/(V A Z)比值呈明显的正线性关系(R=0．9488^***)。这些结果充分证明依赖与叶黄素循环的热耗散是茶树叶片光合器官防御强光破坏的主要途径。     

Isolation and Characterization of a Xanthophyll Aberrant Mutant of the Green Alga Nannochloropsis oculata

Novel mutants (xan1 and xan2) of the unicellular green alga Nannochloropsis oculata are impaired in xanthophyll biosynthesis, thereby producing aberrant levels of xanthophylls. High-performance liquid chromatography (HPLC) analysis revealed that the xan1 and xan2 mutants have double the violaxanthin (V) content, but have significantly decreased lutein content in their cells compared to the wild type. Furthermore, these mutants contain two to three times more zeaxanthin than the wild type under low light (LL) growth conditions. However, this xanthophyll aberration in N. oculata did not affect the normal growth and the major cellular chemical composition of the xan1 strain. The xanthophyll pool size of the LL-grown mutant was 1.8-fold greater than that of the wild type. Under high light (HL) growth conditions, V content was substantially decreased in both the mutant and wild types because of the epoxidation state of the xanthophylls. Under LL growth conditions, the deepoxidation states of the xanthophyll pool sizes were 0.1 and 1.2 in the wild type and the mutant, respectively. However, the deepoxidation states of the xanthophyll pool sizes were 0.78 in the wild type and 0.87 in the mutant under HL growth conditions. We observed that the level of one of the commercially important xanthophylls, zeaxanthin, was higher in the mutant than in the wild type under all culture conditions. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin.     

Photosynthetic acclimation of Pinus taeda (loblolly pine) to long-term growth in elevated pCO2 (FACE)

Growth in elevated pCO₂ generally leads to a stimulation of net CO₂ uptake rate. However, with long‐term growth the magnitude of this stimulation is often reduced. This phenomenon, termed acclimation, has been largely attributed to a loss of Rubisco (ribulose 1,5 bisphosphate carboxylase). The mechanism by which Rubisco content declines with long‐term growth is not certain. There is evidence for a sugar‐mediated, selective down‐regulation of Rubisco protein and also for a non‐selective loss of total leaf nitrogen, which impacts Rubisco levels indirectly. Over a season, and including needles at different developmental stages, we investigated these two potential mechanisms in well‐developed Pinus taeda grown for approximately 2·5 years in elevated (56 Pa) pCO₂ using free air CO₂ enrichment technology. Photosynthetic acclimation, as manifested by a decrease in the activity of Rubisco measured both in vivo (? 25%, via gas exchange) and in vitro (? 35%, via enzyme assays), was observed with growth in elevated pCO₂. This acclimation was observed in one‐year‐old needles but not in current‐year needles. Needles exhibiting acclimation had reduced levels of Lsu Rubisco (? 25%) and an increased foliar carbohydrate content (+ 30%) but showed no evidence of a decrease in needle nitrogen or total protein content. These data support the concept that photosynthetic acclimation in elevated pCO₂ is caused by a selective down‐regulation of Rubisco.     

Two different strategies for light utilization in photosynthesis in relation to growth and cold acclimation

Seedlings of Lodgepole pine (Pinus contorta L.) and winter wheat (Triticum aestivum L. cv. Monopol) were cold acclimated under controlled conditions to induce frost hardiness. Lodgepole pine responded to cold acclimation by partial inhibition of photosynthesis with an associated partial loss of photosystem II reaction centres, and a reduction in needle chlorophyll content. This was accompanied by a low daily carbon gain, and the development of a high and sustained capacity for non‐photochemical quenching of absorbed light. This sustained dissipation of absorbed light as heat correlated with an increased de‐epoxidation of the xanthophyll cycle pigments forming the quenching forms antheraxanthin and zeaxanthin. In addition, the PsbS protein known to bind chlorophyll and the xanthophyll cycle pigments increased strongly during cold acclimation of pine. In contrast, winter wheat maintained high photosynthetic rates, showed no loss of chlorophyll content per leaf area, and exhibited a high daily carbon gain and a minimal non‐photochemical quenching after cold acclimation. In accordance, cold acclimation of wheat neither increased the de‐epoxidation of the xanthophylls nor the content of the PsbS protein. These different responses of photosynthesis to cold acclimation are correlated with pine, reducing its need for assimilates when entering dormancy associated with termination of primary growth, whereas winter wheat maintains a high need for assimilates as it continues to grow and develop throughout the cold‐acclimation period. It appears that without evolving a sustained ability for controlled dissipation of absorbed light as heat throughout the winter, winter green conifers would not have managed to adapt and establish themselves so successfully in the cold climatic zones of the northern hemisphere.     

Thermal Dissipation of Excess Light inArabidopsis Leaves is Inhibited after Gamma-irradiation

To elucidate the effect of ionizing radiation on the non-photochemical quenching (NPQ) oir chlorophyll fluorescence, we analyzed the buildup and release of NPQ inArabidopsis wild-type (WT) andnpq1- 2 mutant plants after gamma-irradiation. Thenpqi- 2 mutant cannot normally induce the buildup of NPQ by a mutation in the violaxamthin de-epoxidase gene. A dose of 50 Gy h for 4 h significantly suppressed such buildup in the mutant and, more noticeably, in the WT. Both the initial rise and maximum level of NPQ were gradually inhibited after gamma-irradiation. In contrast, the release of NPQ and the maximum photochemical efficiency (Fv/Fm) of Photosystem II were largely unaffected in either genotype. This inhibition of NPQ buildup could be partly attributable to a significant decrease in the content of carotenoids, including xanthophyll pigments. Moreover, inhibition that was dependent on the xanthophyll cycle substantially enhanced the sensitivity of irradiated leaves to a photoinhibitory illumination of 800 |imol photons m 2 s"1. The difference in Fv/Fm values between the WT andnpq1- 2 under that photoinhibitory level of illumination was much smaller in the irradiated leaves than in the control. However, NPQ inhibition did not cause a significant difference in efficiency between WT and mutant when both were treated with UV-B irradiance of 2.4 W m 2. Therefore, we suggest that a significant decrease in carotenoid content after gamma-irradiation should partially contribute to the enhanced sensitivity of irradiated plants, at least to high-ligtil photoinhibition. This is accomplished by suppressing the thermal dissipation of excess light absorbed by photosynthetic pigments.     

Strategies providing success in a variable habitat: III. Dynamic control of photosynthesis in Cladophora glomerata

Diurnal patterns of photosynthesis were studied in July and April populations of Cladophora glomerata (L.) Kütz. from open and from shaded sites. Summer samples exposed to full sunlight showed decreased efficiency of open photosystem II at noon, and only slight differences were found between samples that had grown at open or at shaded sites. Electron transport rate was limited at highest fluence rates in shade plants, and non‐photochemical quenching (NPQ) revealed faster regulation in samples from open sites. Daily course of de‐epoxidation was not linearly correlated with the course of NPQ. The comparison of samples from open and from shaded sites revealed a higher capacity of thermal energy dissipation and an increase in the total amount of xanthophyll‐cycle pigments (21%) in samples from open sites. In April, down‐regulation of the efficiency of open photosystem II was related to lower water temperature, and hence, increased excitation pressure. In April the pool size of xanthophyll‐cycle pigments was increased by 21% in comparison with summer and suggested higher levels of thermal energy dissipation via de‐epoxidized xanthophylls. In both, summer and spring the amount of xanthophyll‐cycle pigments was 20% higher in samples from open sites. Acclimation of C. glomerata to growth light conditions was further shown by experimental induction of NPQ, indicating NPQ increases of 23%, and increases of 77% in the reversible component of NPQ in open site samples. The effect of temperature on photosynthetic rate was non‐linear, and different optimum temperatures of electron transport rate and oxygen evolution were exhibited.     

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non‐photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress‐induced photoinhibition and photooxidation, antisense‐mediated suppression of zeaxanthin epoxidase transgenic plants and the wild‐type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^??) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress‐induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.     

The function of chloroplastic NAD(P)H dehydrogenase in tobacco during chilling stress under low irradiance

The function of chloroplastic NAD(P)H dehydrogenase (NDH) was examined by comparing a tobacco transformant (DeltandhB) in which the ndhB gene had been disrupted with its wild type, upon exposure to chilling temperature (4 degrees C) under low irradiance (100 micro mol m(-2) s(-1) PFD). During the chilling stress, the maximum photochemical efficiency of PSII (F(v)/F(m)) decreased markedly in both the wild type and DeltandhB. However, both F(v)/F(m) and P700(+), as well as the PSII-driven electron transport rate (ETR), in DeltandhB were lower than that in the wild type, implying that NDH-dependent cyclic electron flow around PSI functioned to protect the photosynthetic apparatus from chilling stress under low irradiance. Under the stress, non-photochemical quenching (NPQ), particularly the fast relaxing NPQ component (qf) and the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), were distinguishable in DeltandhB from those in the wild type. The lower NPQ in DeltandhB might be related to an inefficient proton gradient across thylakoid membranes (DeltapH) because of lacking an NDH-dependent cyclic electron flow around PSI at chilling temperature under low irradiance.     

Cold acclimation of SnRK2.2 kinases mutant Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, plant‐specific serine/threonine kinases (SnRK2 kinases) play a central role in sulfur metabolism. However, their role in environmental stress has not been clearly understood. Cold stress is one of the most important factors that limit the growth, productivity, and development of photosynthetic organisms. In this report, the effect of cold stress on some physiological parameters were investigated in SnRK2.2 mutant and wild type C. reinhardtii culture. Our results showed that cold stress had significantly enhanced lipid peroxidation rate in wild type, while no significant change was observed in SnRK2.2 mutant culture. Our data also indicated a decline in Rubisco protein amount of wild type culture under low temperature exposure. Low temperature reduced glutathione reductase (GR) activity in the wild type, while GR activity was enhanced in SnRK2.2 mutant. This result indicated the potential of SnRK2 kinase in cold acclimation process.     

Ferredoxin-quinone reductase benefits cyclic electron flow around photosystem 1 in tobacco leaves upon exposure to chilling stress under low irradiance

The function of chloroplast ferredoxin quinone reductase (FQR)-dependent flow was examined by comparing a wild type tobacco and a tobacco transformant (ΔndhB) in which the ndhB gene had been disrupted with their antimycin A (AA)-fed leaves upon exposure to chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹ photon flux density). During the chilling stress, the maximum photochemical efficiency of photosystem (PS) 2 (F_v/F_m) decreased markedly in both the controls and AA-fed leaves, and P700⁺ was also lower in AA-fed leaves than in the controls, implying that FQR-dependent cyclic electron flow around PS1 functioned to protect the photosynthetic apparatus from chilling stress under low irradiance. Under such stress, non-photochemical quenching (NPQ), particularly the fast relaxing NPQ component (q_f) and the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), formed the difference between AA-fed leaves and controls. The lower NPQ in AA-fed leaves might be related to an inefficient proton gradient across thylakoid membranes (ΔpH) because of inhibiting an FQR-dependent cyclic electron flow around PS1 at chilling temperature under low irradiance.     

PHOTOPROTECTION OF SEA‐ICE MICROALGAL COMMUNITIES FROM THE EAST ANTARCTIC PACK ICE1

All photosynthetic organisms endeavor to balance energy supply with demand. For sea‐ice diatoms, as with all marine photoautotrophs, light is the most important factor for determining growth and carbon‐fixation rates. Light varies from extremely low to often relatively high irradiances within the sea‐ice environment, meaning that sea‐ice algae require moderate physiological plasticity that is necessary for rapid light acclimation and photoprotection. This study investigated photoprotective mechanisms employed by bottom Antarctic sea‐ice algae in response to relatively high irradiances to understand how they acclimate to the environmental conditions presented during early spring, as the light climate begins to intensify and snow and sea‐ice thinning commences. The sea‐ice microalgae displayed high photosynthetic plasticity to increased irradiance, with a rapid decline in photochemical efficiency that was completely reversible when placed under low light. Similarly, the photoprotective xanthophyll pigment diatoxanthin (Dt) was immediately activated but reversed during recovery under low light. The xanthophyll inhibitor dithiothreitol (DTT) and state transition inhibitor sodium fluoride (NaF) were used in under‐ice in situ incubations and revealed that nonphotochemical quenching (NPQ) via xanthophyll‐cycle activation was the preferred method for light acclimation and photoprotection by bottom sea‐ice algae. This study showed that bottom sea‐ice algae from the east Antarctic possess a high level of plasticity in their light‐acclimation capabilities and identified the xanthophyll cycle as a critical mechanism in photoprotection and the preferred means by which sea‐ice diatoms regulate energy flow to PSII.     

A nonphotochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity

Higher-plant chloroplasts alter the distribution of absorbed radiant energy between photosynthesis and heat formation in response to changing illumination level or environmental stress. Fluorescence imaging was used to screen 62 yellow-green T-DNA insertion mutant lines of Arabidopsis thaliana (L.) Heynh. for reduced photoprotective nonphotochemical quenching (NPQ) capacity. Pulse-modulation fluorometry was employed to characterize one line (denoted Lsr1⁻) that exhibited an approximately 50% reduction in NPQ compared to the wild type (WT). The loss in NPQ capacity was associated with the ΔpH-dependent phase of quenching (qE). Under the growth conditions employed, pigment composition and levels of the six photosystem-II light-harvesting chlorophyll a/b proteins were identical in mutant and WT. Changes in the in-vivo levels of the xanthophyll pigments violaxanthin, antheraxanthin, and zeaxanthin in excess light were the same for mutant and WT. However, use of the violaxanthin de-epoxidase inhibitor dithiothreitol indicated that a zeaxanthin-dependent component of NPQ was specifically reduced in the mutant. The mutant exhibited diminished suppression of minimum fluorescence yield (F _o ) in intense light suggesting an altered threshold in the mechanism of response to light stress in the mutant. The NPQ-deficient phenotype was meiotically transmissible as a semidominant trait and mapped near marker T27K12 on chromosome 1. The results suggest that the mutant is defective in sensing the transthylakoid ΔpH that reports exposure to excessive illumination. Received: 26 May 1999 / Accepted: 17 June 1999     

Xanthophyll cycle components and capacity for non-radiative energy dissipation in sun and shade leaves ofLigustrum ovalifolium exposed to conditions limiting photosynthesis

The relationships between photosynthetic efficiency, non-radiative energy dissipation and carotenoid composition were studied in leaves ofLigustrum ovalifolium developed either under full sunlight or in the shade. Sun leaves contained a much greater pool of xanthophyll cycle components than shade leaves. The rate of non-radiative energy dissipation, measured as non-photochemical fluorescence quenching (NPQ), was strictly related to the deepoxidation state (DPS) of xanthophyll cycle components in both sun and shade leaves, indicating that zeaxanthin (Z) and antheraxanthin (A) are involved in the development of NPQ. Under extreme conditions of excessive energy, sun leaves showed higher maximum DPS than shade leaves. Therefore, sun leaves contained not only a greater pool of xanthophyll cycle components but also a higher proportion of violaxanthin (V) actually photoconvertible to A and Z, compared to shade leaves. Both these effects contributed to the higher NPQ in sun versus shade leaves. The amount of photoconvertible V was strongly related to chla/b ratio and inversely to leaf neoxanthin content. This evidence indicates that the amount of photoconvertible V may be dependent on the degree of thylakoid membrane appression and on the organization of chlorophyll-protein complexes, and possible explanations are discussed. Exposure to chilling temperatures caused a strong decline in the photon yield of photosynthesis and in the intrinsic efficiency of PS II photochemistry in sun leaves, but little effects in shade leaves. These effects were accompanied by increases in the pool of xanthophyll cycle components and in DPS, more pronounced in sun than in shade leaves. This corroborates the view that Z and A may play a photoprotective role under unfavorable conditions. In addition to the xanthophyll-related non-radiative energy dissipation, a slow relaxing component of NPQ, independent from A and Z concentrations, has been found in leaves exposed to low temperature and high light. This quenching component may be attributed either to other regulatory mechanism of PS II efficiency or to photoinactivation.Research supported by National Research Council of Italy, Special Project RAISA, Sub-Project 2, Paper N. 1587.     

Analysis of xanthophyll cycle carotenoids and chlorophyll fluorescence in light intensity-dependent chlorophyll-deficient mutants of wheat and barley

Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 mol photons·m^–2 s^–1 incident light). Mutants grown under low light possessed lower levels of total Chls and carotenoids per unit leaf area compared to wild type plants, but the relative proportions of the two did not vary markedly between strains. In contrast, mutants grown under high light had much lower levels of Chl, leading to markedly greater carotenoid to Chl ratios in the mutants when compared to wild type. Under low light conditions the carotenoids of the xanthophyll cycle comprised approximately 15% of the total carotenoids in all strains; under high light the xanthophyll cycle pool increased to over 30% of the total carotenoids in wild type plants and to over 50% of the total carotenoids in the three mutant strains. Whereas the xanthophyll cycle remained fairly epoxidized in all plants grown under low light, plants grown under high light exhibited a considerable degree of conversion of the xanthophyll cycle into antheraxanthin and zeaxanthin during the diurnal cycle, with almost complete conversion (over 90%) occurring only in the mutants. 50 to 95% of the xanthophyll cycle was retained as antheraxanthin and zeaxanthin overnight in these mutants which also exhibited sustained depressions in PS II photochemical efficiency (F_v/F_m), which may have resulted from a sustained high level of photoprotective energy dissipation activity. The relatively larger xanthophyll cycle pool in the Chl b-deficient mutant could result in part from the reported concentration of the xanthophyll cycle in the inner antenna complexes, given that the Chl b-deficient mutants are deficient in the peripheral LHC-II complexes.Abbreviations A antheraxanthin - Chl chlorophyll - F_o and F_m minimal yield (at open PS II reaction centers) and maximal yield (at closed centers) of chlorophyll fluorescence in darkness - F level of fluorescence during illumination with photosynthetically active radiation - F_m maximal yield (at closed centers) of chlorophyll fluorescence during illumination with photosynthetically active radiation - (F_m–F)/F_m actual efficiency of PS II during illumination with photosynthetically active radiation - F_v/F_m+(F_m–F_o)/F_m intrinsic efficiency of PS II in darkness - LHC_II light-harvesting chlorophyll-protein complex of Photosystem II - PFD photon flux density (between 400 and 700 nm) - PS I Photosystem I - PS II Photosystem II - V violaxanthin - Z zeaxanthin     

Distribution of N, Rubisco and photosynthesis in Pinus pinaster and acclimation to light

The light–nitrogen hypothesis suggests canopy photosynthesis is maximized when there is a positive relationship between irradiance received by foliage, its nitrogen content (per unit area N_area), and maximum rate of photosynthesis (A_max). Relationships among relative irradiance and N_area, allocation of nitrogen within the photosynthetic apparatus to Rubisco and chlorophyll, and A_max were examined in Pinus pinaster Ait. needles up to 6 years of age. Measurements were made before bud break in August 1998, and in May 1999 after the first ‘winter’ rains. In August, N_area in P. pinaster needles decreased from 5·1 to 5·7 g m^?2 in sunlit 1‐year‐old needles to 2·3 g m^?2 in shaded 6‐year‐old needles. In May, N_area was 5–40% less but spatial trends were the same. At both sampling dates, A_max was less in old shaded needles compared with young sunlit needles, and was thus consistent with the light–nitrogen hypothesis. Relationships between N_area and A_max were positive at both dates yet varied in strength and form. Allocation of nitrogen within the photosynthetic apparatus was qualitatively consistent with acclimation to light (i.e. Rubisco/Chl decreased with shading), but quantitatively suboptimal with respect to photosynthesis owing to consistent over‐investment in Rubisco. This over‐investment increased with height in the canopy and was greater in May than in August.     

Effects of ultraviolet (UV) exclusion on the seasonal concentration of photosynthetic and UV-screening pigments in Scots pine needles

The effects of ultraviolet (UV) radiation on the photosynthetic and UV‐screening pigments in needles of Scots pine (Pinus sylvestris L.) saplings were studied in a UV‐exclusion field chamber experiment in northern Finland (67°N) during 2001–2002. The chambers held filters that excluded both UVB and UVA, only UVB, transmitted all UV, or lacked filters. Analyses of control needles (no filter and polyethene filter) showed that the first changes to occur in spring (end of April) was an abrupt increase in the epoxidation state (EPS) of the xanthophyll cycle pigments, likely in relation with the beginning of the photosynthetic activity. The concentration of chlorophyll, lutein, neoxanthin, α‐carotene, β‐carotene, and the size of the xanthophyll cycle pool (violaxanthin+antheraxanthin+zeaxanthin=VAZ) changed only later when needles reached their summer photosynthesis state. Exclusion of UV radiation significantly affected the xanthophyll cycle but not the other photosynthetic pigments analysed. Interestingly, the effects on xanthophylls were dependent on the sampling date. Under UVA/B‐exclusion, the EPS was increased and VAZ pool size was unchanged in April, whereas EPS remained unchanged and the VAZ pool size was reduced in May and June. The existence of two sustained and active antenna modes during winter and summer could be an explanation for the specific UV‐exclusion effect in the different season. A high‐performance liquid chromatography analysis of soluble phenolics showed that the exclusion of UVA/B radiation caused a significant effect on five compounds out of 46 studied, without affecting the concentration of the total soluble phenolics. Under UVA/B‐exclusion, the concentration of three of them (secoisolariciresinol‐glucopyranoside, two unknown) was reduced while the concentration of dicoumaroyl‐astragalin and pinosylvin monomethylether was increased compared with both controls separately. In general, the exclusion of UVA/B caused a stronger effect than the exclusion of UVB on both photosynthetic and UV screening pigments. The effects of UV radiation on xanthophyll cycle pigments were season‐specific and detectable only under stressful spring conditions (freezing temperatures and high irradiance due to snow reflection). The effect on the xanthophyll cycle could be a direct consequence of UV treatments, or an indirect consequence of the changed flavonoid composition, or a combination of both.     

Mutation design of a thermophilic Rubisco based on three‐dimensional structure enhances its activity at ambient temperature

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk‐Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one‐eighth the activity at ambient temperature. We have tried to improve the activity of Tk‐Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild‐type enzyme both in vitro and in vivo. Here, we designed new Tk‐Rubisco mutants based on its three‐dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild‐type enzyme. The crystal structure of the Tk‐Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk‐Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8‐T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8‐T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two‐fold higher specific growth rates compared to that of a strain harboring the wild‐type enzyme at ambient temperature. Proteins 2016; 84:1339–1346. © 2016 Wiley Periodicals, Inc.