Photosynthetic pigments and gas exchange during ex vitro acclimation of tobacco plants as affected by CO2 supply and abscisic acid

Nicotiana tabacum L. plantlets were grown in glass vessels or in Magenta boxes with better CO₂ supply. To improve the ex vitro transfer we tested application of abscisic acid and elevated CO₂ concentration. In the first two weeks after transfer, net photosynthetic rate, chlorophyll a+b content, and Chl a/b ratio were higher, and content of xanthophyll cycle pigments lower in M-plants than in G-plants, but during further growth the differences almost disappeared. ABA application alleviated the risk of wilting because it decreased stomatal conductance. The effect of ABA was enhanced under CE (28 days after transfer). In situ, P_N was always higher at CE than at CA, but when measured under CA, positive effect of CE was found 2 and 16 days after transfer in M-plants and only 16 days after transfer in G-plants. Slightly increased Chl a content was found in all ABA-treated plants, and in M-plants grown under CE. The content of xanthophyll cycle pigments was lower under CE compared to CA, and the lowest one was found in ABA-treated M-plants grown under CE. On the contrary, the degree of their deepoxidation (DEPS) was slightly higher in plants grown under CE. No significant effects of ABA-treatment or growth under CE on fluorescence kinetic parameters were found and inconsistent effects on photochemical activities. The photochemical efficiency of PS2 (variable to maximum fluorescence ratio, F_v/F_m) after ex vitro transfer was similar to that in in vitro grown plants. This together with the values of DEPS indicated that no photodamage during ex vitro transfer occurred. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthetic Pigments and Gas Exchange of in vitro Grown Tobacco Plants as Affected by CO2 Supply

Contents and functioning of photosynthetic pigments and gas exchange of Nicotiana tabacum L. leaves were studied in platlets cultivated in vitro under different CO₂ supply. The plantlets were grown for six weeks either in glass vessels tightly closed with aluminium foil (G-plants) or in polycarbonate Magenta GA-7 vessels covered with closures with microporous vents (M-plants). M-plants (better supplied with CO₂) had higher contents of chlorophyll (Chl) a. Chl b. and β-carotene, higher photochemical activities of photosystem 2 and whole electron transport chain, and lower contents of xanthophyll cycle pigments. Differences in Chl a fluorescence kinetic parameters between G-plants and M-plants were not statistically significant. M-plants had higher net photosynthetic rate, and lower transpiration rate and stomatal conductance than G-plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of abscisic acid on photosynthetic parameters during <Emphasis Type="Italic">ex vitro</Emphasis> transfer of micropropagated tobacco plantlets

The aim of this research was to determine whether exogenous abscisic acid (ABA) applied immediately after ex vitro transfer of in vitro grown plants can improve their acclimatization. Tobacco (Nicotiana tabacum L.) plantlets were transferred into pots with Perlite initially moistened either by water or 50 μM ABA solution and they were grown under low (LI) or high (HI) irradiance of 150 and 700 μmol m⁻² s⁻¹, respectively. Endogenous content of ABA in tobacco leaves increased considerably after ABA application and even more in plants grown under HI. Stomatal conductance, transpiration rate and net photosynthetic rate decreased considerably 1 d after ex vitro transfer and increased thereafter. The gas exchange parameters were further decreased by ABA application and so wilting of these plants was limited. Chlorophyll (a+b) and β-carotene contents were higher in ABA-treated plants, but the content of xanthophyll cycle pigments was not increased. However, the degree of xanthophyll cycle pigments deepoxidation was decreased what also suggested less stress in ABA-treated plants. No dramatic changes in most chlorophyll a fluorescence parameters after ex vitro transfer suggested that the plants did not suffer from restriction of electron transport or photosystem damage.     

Impact of in vitro Cultivation Conditions on Stress Responses and on Changes in Thylakoid Membrane Proteins and Pigments of Tobacco during ex vitro Acclimation

Four physiologically and phenotypically diversified tobacco (Nicotiana tabacum L. cv. Samsun) plantlet variants had been generated by cultivation on media either lacking or containing sucrose (0 and 3 %, m/v) under two different photon flux densities (PFD), 50 µmol m^–2 s^–1 (LL) and 200 µmol m^–2 s^–1 (HL). Plantlets were transferred into soil without any pre-acclimation and grown either under PFD of 200 µmol m^–2 s^–1 or 700 µmol m^–2 s^–1. Sucrose feeding in vitro resulted in reduced degree and duration of wilting after transfer. The highest readiness for ex vitro acclimation was found in 3 % HL plants, in which changes of photosynthetic apparatus and stress responses were the smallest. On the contrary, the steepest decline of Fv/Fm ratio on the first day after transplantation, doubled chlorophyll content and almost tripled D1/LHC 2 ratio after 7 d of ex vitro growth under 700 µmol m^–2 s^–1 characterized 0 % HL plants, which had suffered chronic photoinhibition in vitro. Remarkably high abscisic acid content at the end of in vitro cultivation and during acclimation as well as increased synthesis of both D1 and LHC 2 proteins even at the end of analyzed acclimation period were found only in 0 % LL plants. Increase of D1/LHC 2 ratio and chlorophyll contents demonstrate that in vitro developed leaves of all plant variants are able to acclimate to new environment. The most surprising result in the whole study is the drop of D1 protein synthesis in all plants on the 3^rd day. Five times decline of photoprotection level of xanthophylls in plants after ex vitro transfer into the same PFD showed stress character of in vitro cultures.     

The impact of <Emphasis Type="Italic">trans</Emphasis>-zeatin <Emphasis Type="Italic">O</Emphasis>-glucosyltransferase gene over-expression in tobacco on pigment content and gas exchange

The responses of tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under constitutive or senescence-inducible promoter (35S:ZOG1 and SAG12:ZOG1) and of wild type (WT) plants to water stress and subsequent rehydration were compared. In plants sufficiently supplied with water, both transgenics have higher net photosynthetic rate (P_N) in upper and middle leaves and higher stomatal conductance (g_s) in middle leaves than WT. Water use efficiency (WUE = P_N/E) was higher in both transgenics than in WT. During prolonged water stress, both P_N and E declined to a similar extent in both transgenics and WT plants. However, 7 d after rehydration P_N in SAG:ZOG (upper and middle leaves) and 35S:ZOG (upper leaves) was higher than that in WT plants. Increased content of endogenous CKs in 35S:ZOG plants did not prevent their response to ABA application and the results obtained did not support concept of CK antagonism of ABA-induced stomatal closure. The chlorophyll (Chl) a+b content was mostly higher in both transgenics than in WT. During water stress and subsequent rehydration it remained unchanged in upper leaves, decreased slightly in middle leaves only of WT, while rapidly in lower leaves. Total degradation of Chl, carotenoids and xanthophyll cycle pigments (XCP) was found under severe water stress in lower leaves. Carotenoid and XCP contents in middle and upper leaves mostly increased during development of water stress and decreased after rehydration. While β-carotene content was mostly higher in WT, neoxanthin content was higher in transgenics especially in 35S:ZOG under severe stress and after rehydration. The higher content of XCP and degree of their deepoxidation were usually found in upper and middle leaves than in lower leaves with exception of SAG:ZOG plants during mild water stress.     

Stomatal Morphology during Acclimatization of Tobacco Plantlets to ex vitro Conditions

Image analysis was used in studying stomatal morphology during acclimatization of tobacco plantlets to ex vitro conditions, 45 d after transfer leaf area was 15 times, and total number of stomata per leaf four times increased. During acclimatization stomatal density was decreased considerably on both leaf sides, and was compensated by an increase in stomatal sizes, e.g., in stomatal length and in stomatal area (both guard cells and pore). Elongation of stomata was increased indicating that the originally circular stomata of in vitro plantlets were changed into elliptical ones in ex vitro acclimatized plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthetic Performance of Ginkgo biloba L. Grown Under High and Low Irradiance

Diurnal variation in net photosynthetic rate (P _N) of three-year-old plants of Ginkgo biloba was studied under open, O (receiving full sunlight), net-shade, NS (40 % of photosynthetically active radiation, PAR), or greenhouse, G (25 % PAR) conditions. In all three conditions, P _N was higher in morning along with stomatal conductance (g _s), and intercellular CO₂ concentration (C _i), while leaf temperature and vapour pressure deficit were low. The O-plants exhibited a typical decline in P _N during midday, which was not observed in NS-plants. This indicated a possible photoinhibition in O-plants as the ratio of variable to maximum fluorescence (F_v/F_m) and photosystem 2 (PS2) yield (_PS2) values were higher in the NS- and G-plants. On the contrary, stomatal density and index, chlorophyll a/b ratio, leaf thickness, and density of mesophyll cells were greater in O-plants. Further, higher P _N throughout the day along with higher relative growth rate under NS as compared to O and G suggested the better efficiency of Ginkgo plants under NS conditions. Therefore, this plant species could be grown at 40 % irradiance to meet the ever-increasing demand of leaf and also to increase its export potential.     

Water relations and photosynthesis in Cucumis sativus L. leaves under salt stress

Hydroponically grown cucumber plants were exposed to 14-d period of salinity (0, 50, 100 mM NaCl). NaCl caused reduction in the relative water content in the leaves. The Na⁺ content increased and the K⁺ content decreased. The net photosynthetic rate, stomatal conductance and transpiration rate were markedly decreased by all of the salt treatments. Salinity decreased also the maximum quantum efficiency of photosystem 2 (PS 2) determined as the variable to maximum fluorescence ratio, the photochemical quantum yield of PS 2 and the photochemical fluorescence quenching, while the non-photochemical quenching increased. Above results indicate that NaCl affects photosynthesis through both stomata closure and non-stomatal factors.     

Different Relationship Between Electron Transport and CO2 Assimilation in two Zea mays Cultivars as Influenced by Increasing Irradiance

Gas exchange and fluorescence parameters were measured simultaneously in two Zea mays L. cultivars (Liri and 121C D8) to assess the relationship between the quantum yield of electron transport (_PS2) and the quantum yield of CO₂ assimilation (_CO2) in response to photosynthetic photon flux density (PPFD). The cv. Liri was grown under controlled environmental conditions in a climate chamber (CC) while cv. 121C D8 was grown in CC as well as outdoors (OT). By exposing the two maize cultivars grown in CC to an increasing PPFD, higher photosynthetic and photochemical rates were evidenced in cv. Liri than in cv. 121C D8. In Liri plants the _PS2/_CO2 ratio increased progressively up to 27 with increasing PPFD. This suggests that the reductive power was more utilised in non-assimilatory processes than in CO₂ assimilation at high PPFD. On the contrary, by exposing 121C D8 plants to increasing PPFD, _PS2/_CO2 was fairly constant (around 11–13), indicating that the electron transport rate was tightly down regulated by CO₂ assimilation. Although no significant differences were found between _PS2/_CO2 of the 121C D8 maize grown under CC and OT by exposing them to high PPFD, the photosynthetic rate and photochemical rates were higher in OT maize plants.     

The Role of Abscisic Acid in Acclimation of Plants Cultivated in vitro to ex vitro Conditions

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of water stress on leaf photosynthetic characteristics in wheat cultivars differing in their susceptibility to drought

A gradual reduction in leaf water potential (Ψ_leaf), net photosynthetic rate (P _N), stomatal conductance, and transpiration rate was observed in two drought tolerant (C 306 and K 8027) and two susceptible (RW 893 and 899) genotypes subjected to water stress. The extent of reduction was lower in K 8027 and C 306 and higher in RW 893 and RW 899. Rewatering the plants after 5 d of stress restored P _N and other gas exchange traits in all four cultivars. Water stress had no significant effect on variable to maximum fluorescence ratio (F_v/F_m) indicating that water stress had no effect on primary photochemistry of photosystem 2 (PS2). However, water stress reduced the efficiency of excitation energy transfer (F′_v/F′_m) and the quantum yield of electron transport (Φ_PS2). The reduction was more pronounced in susceptible cultivars. Water stress had no significant effect on photochemical quenching, however, the non-photochemical quenching increased by water stress.     

Photosynthesis in different types of transgenic tobacco plants with elevated cytokinin content

Photosynthetic parameters were compared in three types of transgenic tobacco plants: ipt-transgenic plants with slightly elevated endogenous cytokinin (CK) content, Pssu-ipt-transgenic plants with markedly increased CK content, and zmp-transgenic plants with slightly elevated CK content accompanied by elevated auxin content. Slightly increased CK content promoted net photosynthetic rate (P_N) in both ipt- and zmp-transgenic plants, and chlorophyll (Chl) and carotenoid contents in zmp-transgenic plants. Morphology, growth characteristics, stomatal conductance, and parameters of Chl a fluorescence kinetics were similar in control and transgenic plants with slightly higher CK content. No significant effect of increased level of endogenous auxin (indole-3-acetic acid) on development of zmp-transgenic plants and measured parameters was found. Pssu-ipt-transgenic plants with highly increased CK content revealed suppressed root development, wilting of plants, and depression of P_N and stomatal conductance; however, Chl content was slightly increased and parameters of Chl a fluorescence kinetics did not indicate damage to photosynthetic apparatus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Benzylaminopurine on Rehydration of Bean Plants after Water Stress

The possibility of improving the recovery of plant photosynthesis after water stress by cytokinin-induced stimulation of stomatal opening or delay of leaf senescence was tested. The 6-benzylaminopurine (BAP) in concentrations 1 and 10 M was applied to the substrate (sand + nutrient solution) or sprayed on primary leaves of 14-d-old Phaseolus vulgaris L. plants sufficiently supplied with water or water-stressed for 4 d. The later ones having relative water content decreased to 69 % were fully rehydrated during the following three days. Parameters of photosynthesis and water relations were measured in primary leaves of 7-, 10-, 14-, and 17-d-old plants. Application of 1 M BAP slightly delayed leaf senescence: in 17-d-old control plants, net photosynthetic rate (P_N) and chlorophyll (Chl) content, and when sprayed on leaves also some of Chl a fluorescence kinetic parameters of BAP-treated leaves were slightly higher than those of untreated leaves. Both types of application of 1 M BAP slightly improved recovery of plants during rehydration after water stress in terms of increased g_ad, g_ab and P_N, i.e., parameters which were markedly decreased by mild water stress. However, contents of Chl a, Chl b and carotenoids and parameters of Chl a fluorescence kinetic were not markedly affected by mild water stress and after rehydration were not stimulated by 1 M BAP. 10 M BAP had mostly negative effects on the parameters measured.     

Production of Doubled Haploids from Androgenic Embryoids and Plantlets of Tobacco

In four experiments, the chromosome doubling effect of acenaphthene vapour was tested on androgenic embryoids and/or plantlets of tobacco. The maximum rate of plants with doubled chromosome numbers (41.3 % diploids plus 2.7 % tetraploids) was recorded when approx. 100 mg acenaphthene in small and sterilized cheese-cloth bags were hanged for 72 h, into the culture jars (6.0 × 11.5 cm), containing 4 – 6 androgenic plantlets newly transferred from culture tubes. Acenaphthene treatments performed at the earlier stages of culture resulted in lower percentages (from 0.0 to 6.3 %). This revised version was published online in July 2006 with corrections to the Cover Date.     

Photosynthetic Responses of Tropical Trees to Short-Term Exposure to Ozone

Saplings of the tropical trees Tibouchina pulchra (Cham.) Cogn., Caesalpinia echinata Lam., and Psidium guajava L. cv. Paluma were exposed in open-top chambers with charcoal filtered air and measurements of gas exchange and chlorophyll fluorescence were made before (t₁) and after exposure to non-filtered air plus O₃ (t₂), simulating 6-h peaks of O₃ similar to those observed in São Paulo city (SE Brazil, reaching an AOT40 of 641 nmol mol^–1). After the fumigation, the net photosynthetic rate, stomatal conductance, transpiration rate, and F_v/F_m were reduced (p<0.05) for the three species. C. echinata was the most sensitive species and P. guajava cv. Paluma the most resistant.     

Leaf Structure of Tobacco In Vitro Grown Plantlets as Affected by Saccharose and Irradiance

Tobacco plantlets were cultured in vitro under high (200 µmol m^–2 s^–1) or low (60 µmol m^–2 s^–1) irradiance with or without saccharose in the medium. Light microscopy and image analysis were used to evaluate the effect of these culture conditions on leaf anatomy. Addition of saccharose resulted in thicker leaves (all leaf layers) and larger mesophyll cells under both growth irradiances. Various irradiance affected leaf anatomy differently when plantlets had been cultivated in presence or absence of saccharose in the medium. While under high irradiance in presence of saccharose leaf thickness and number of chloroplasts per cell section were increased, plantlets grown under high irradiance in absence of saccharose had thinner leaves and less chloroplasts per cell section. The changes were more pronounced in palisade parenchyma layer.     

Photosynthesis of Hedera Canariensis var. Azorica Variegated Leaves as Affected by Ozone

A differential response to long-term ozone exposures (50 and 100 mm3 m-3) was observed in the green and white areas of variegated leaves of Hedera canariensis var. azorica L. In green tissue the photosynthetic activity was depressed via a stomatal mechanism, and in white regions no effect was observed. Chlorophyll fluorescence parameters remained unchanged in green portions, whereas in the white ones Fm and Fv/Fm significantly diminished following ozone fumigation.     

Relationship Between Photosystem 2 Electron Transport and Photosynthetic CO2 Assimilation Responses to Irradiance in Young Apple Tree Leaves

The responses to irradiance of photosynthetic CO₂ assimilation and photosystem 2 (PS2) electron transport were simultaneously studied by gas exchange and chlorophyll (Chl) fluorescence measurement in two-year-old apple tree leaves (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd). Net photosynthetic rate (P _N) was saturated at photosynthetic photon flux density (PPFD) 600-1 100 (mol m^-2 s^-1, while the PS2 non-cyclic electron transport (P-rate) showed a maximum at PPFD 800 mol m^-2 s^-1. With PPFD increasing, either leaf potential photosynthetic CO₂ assimilation activity (F_d/F_s) and PS2 maximal photochemical activity (F_v/F_m) decreased or the ratio of the inactive PS2 reaction centres (RC) [(F_i – F_o)/(F_m – F_o)] and the slow relaxing non-photochemical Chl fluorescence quenching (q_s) increased from PPFD 1 200 mol m^-2 s^-1, but cyclic electron transport around photosystem 1 (RFp), irradiance induced PS2 RC closure [(F_s – F_o)/F_m – F_o)], and the fast and medium relaxing non-photochemical Chl fluorescence quenching (q_f and q_m) increased remarkably from PPFD 900 (mol m^-2 s^-1. Hence leaf photosynthesis of young apple leaves saturated at PPFD 800 mol m^-2 s^-1 and photoinhibition occurred above PPFD 900 mol m^-2 s^-1. During the photoinhibition at different irradiances, young apple tree leaves could dissipate excess photons mainly by energy quenching and state transition mechanisms at PPFD 900-1 100 mol m^-2 s^-1, but photosynthetic apparatus damage was unavoidable from PPFD 1 200 mol m^-2 s^-1. We propose that Chl fluorescence parameter P-rate is superior to the gas exchange parameter P _N and the Chl fluorescence parameter F_v/F_m as a definition of saturation irradiance and photoinhibition of plant leaves.     

Effects of Reddening of Cotton (Gossypium hirsutum L.) Leaves on Functional Activity of Photosynthetic Apparatus

Strong inhibition of rates of CO₂ assimilation and transpiration, stomatal conductance, and water use efficiency as well as photosystem 2 (PS2) photochemical activity were related to the severity of reddening. The inhibition of photosynthesis in red cotton leaves was due to both decreased photochemical activity and stomatal limitation. Lowered photosynthetic capacity could be one of the main factors of reduced yield in reddening cotton.     

Acclimatization of Micropropagated Plants to Ex Vitro Conditions

The special conditions during in vitro culture result in the formation of plantlets of abnormal morphology, anatomy and physiology. After ex vitro transfer, these plantlets might easily be impaired by sudden changes in environmental conditions, and so need a period of acclimatization to correct the abnormalities. This review is focused upon contemporary information on the changes in leaf structure, water relations and photosynthesis during acclimatization of plantlets to ex vitro conditions. It also describes some ways of improving plant survival and for the speeding up of acclimatization. This revised version was published online in July 2006 with corrections to the Cover Date.