Helena Synková

Cytokinins and water stress

It is almost impossible to find a single process in plant life that is not affected by both stress and hormones directly or indirectly. This minireview is focused on the interactions between water stress and cytokinins (CKs). The attention was paid mainly to changes in endogenous CK content and composition under water stress, involvement of CK in plant responses to water stress mainly in stomatal regulation of gas exchange, water relations of transgenic plants with elevated CK content, and possibilities to ameliorate the negative effects of water stress by application of exogenous CKs.

Production of reactive oxygen species and development of antioxidative systems during in vitro growth and ex vitro transfer

Ex vitro transfer is often stressful for in vitro grown plantlets. Water stress and photoinhibition, often accompanying the acclimatization of in vitro grown plantlets to ex vitro conditions, are probably the main factors promoting production of reactive oxygen species (ROS) and in consequence oxidative stress. The extent of the damaging effects of ROS depends on the effectiveness of the antioxidative systems which include low molecular mass antioxidants (ascorbate, glutathione, tocopherols, carotenoids, phenols) and antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase). This review is focused on ROS production and development of antioxidative system during in vitro growth and their further changes during ex vitro transfer.

Photosynthesis of Transgenic Pssu-ipt Tobacco

Summary The elevated content of endogenous cytokinin (CK), as the result of the introduction of the chimeric P ssu-ipt gene in tobacco, induced changes in growth, water regime and photosynthesis of transgenic grafts and rooted plants grown in a greenhouse. Despite closed stomata remarkable water stress was detected in P ssu-ipt plants. The ABA content was lower than in wild type tobacco, indicative of disturbances in ABA transport or metabolism. The rates of net photosynthesis (measured as CO 2 uptake or O 2 evolution) decreased by more than 50% in P ssu-ipt grafts and by 20% in P ssu-ipt rooted plants, respectively. The partial reactions of the electron transport chain in transgenic P ssu-ipt tobacco were differently influenced: the activity of the reaction centre of PSII was hardly affected; the PS I activity and the intersystem electron transport chain was inhibited up to 70%, particularly in the P ssu-ipt grafts. Although the slight decrease in the potential photochemical efficiency of PSII, expressed as F v /F m , was found in transgenic plants, the actual quantum yields, photochemical efficiencies, and qP under steady-state conditions indicated that transgenic plants were not seriously limited by the redox state of QA. The reaction centre of PSII was well preserved. In transgenic grafts, photophosphorylation capacity was strongly reduced, which could correspond with lower non-photochemical quenching. The above mentioned changes are the results of elevated CK content per se rather than the effect of altered water relations in the plants, caused by the disproportion of shoots and root system in both P ssu-ipt grafts and plants.

Photosynthesis and Activity of Phosphoenolpyruvate carboxylase in Nicotiana tabacum L. Leaves Infected by Potato virus A and Potato virus Y

The influence of viral infection caused by two different potyviruses, Potato virus Y (PVY) and Potato virus A (PVA) on plant metabolism and photosynthetic apparatus of Nicotiana tabacum L. cv. Samsun and cv. Petit Havana SR1 was studied. The main stress was focused on the activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK). The analysis of the presence of viral proteins, enzyme activities, and different photosynthetic parameters showed the time dependent progress of viral infection and NADP-ME and PEPC activities. PVY caused significant response, while PVA affected both tobacco cultivars only slightly. Viral infection, namely PVY, affected more negatively photosynthetic apparatus of cv. Petit Havana SR1 than cv. Samsun.

Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.

High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in Nicotiana tabacum

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions.

Effects of abscisic acid or benzyladenine on pigment contents, chlorophyll fluorescence, and chloroplast ultrastructure during water stress and after rehydration

With the aim to contribute to the elucidation of the role of phytohormones in response of plants to adverse environmental conditions, seedlings of Phaseolus vulgaris, Nicotiana tabacum, Beta vulgaris, and Zea mays were supplied with water, 100 µM abscisic acid (ABA), or 10 µM N6-benzyladenine (BA) immediately before imposition of water stress (WS). In all four species, contents of chlorophylls (Chls) and carotenoids were markedly decreased during WS and after rehydration only in plants pre-treated with water but not in those pre-treated with ABA or BA. Contents of pigments of xanthophyll cycle increased during WS more in plants pre-treated with ABA or BA than in those pre-treated with water, but the degree of their de-epoxidation was highest in the later. Similarly, the efficiency of photosystem 2, determined as variable to maximal Chl fluorescence ratio, was not markedly decreased in bean plants pre-treated with ABA or BA in contrast to those pre-treated with water. The imposed WS was not severe enough to damage chloroplast ultrastructure. However, different changes in a size of starch inclusions were observed. In bean plants, the amount of starch increased considerably in plants pre-treated with water, while it decreased in BA pre-treated plants and no change was found in ABA pre-treated ones. The starch content declined under WS in sugar beet and tobacco plants but only moderate changes were found in ABA or BA pre-treated plants. Thus the application of BA and especially of ABA reduced the negative effects of subsequent WS.

Effect of abscisic acid on photosynthetic parameters during ex vitro 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.

Chlorophyll a fluorescence as a tool for a study of the Potato virus Y effects on photosynthesis of nontransgenic and transgenic Pssu-ipt tobacco

The effect of Potato virus YNTN (PVY) infection upon photosynthesis was analysed in transgenic Pssu-ipt tobacco overproducing endogenous cytokinins in comparison with control, nontransgenic Nicotiana tabacum plants. The course of the infection from the early to the late stage was monitored by measuring of photosynthetic gas exchange and fast chlorophyll (Chl) a fluorescence induction kinetics. Leaf photosynthesis was also analysed using Chl fluorescence imaging (Chl-FI). From the different fluorescence parameters obtained using Chl-FI, the nonphotochemical quenching (NPQ) proved to be the most useful parameter to assess the effect of PVY infection. On the other hand, Chl-FI was found to be inapplicable for any presymptomatic detection of PVY infection in tobacco. The lower accumulation of the virus was found in transgenic plants and corresponded also with the presence of visible symptoms of PVY infection. The net photosynthetic rate (PN), transpiration rate (E), and stomatal conductance (gs) significantly decreased with the progress of the infection in both control plant types and transgenic rooted plants, while transgenic grafts were much less affected. The analysis of the Chl fluorescence transient revealed higher number of silent dissipative reaction centres, higher nonphotochemical dissipation, and significantly lower performance index, PI(abs), in the healthy transgenic grafts. Chl-FI also confirmed significantly higher NPQ in transgenic grafts.

Three-dimensional reconstruction of anomalous chloroplasts in transgenic ipt tobacco

Anomalies in the ultrastructure of chloroplasts, from transgenic ipt tobacco, overproducing endogenous cytokinins (CKs) were studied. Detailed analyses of CKs and their metabolites showed that Pssu-ipt tobacco contained enhanced contents of CKs both in leaves and in isolated chloroplasts. The role of CKs in the formation of anomalous structures is suggested. Pssu-ipt chloroplasts frequently formed the distinct peripheral reticulum with a system of caverns that often involved mitochondria and/or peroxisomes. Large crystalloids, which were found in chloroplasts of Pssu-ipt, occupied up to 16% of chloroplast volume. We suggested that the crystalloids were formed by LHC II aggregates. This was supported by analysis of the fluorescence emission spectra at 77°K, chlorophyll a/b ratio, immunogold staining of the structures, and crystallographic unit size analysis.