D. Haisel

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.

Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content

In order to test the possibility of improving tolerance to heat and drought (alone and in combination) by elevation of the osmoprotectant proline (Pro) content, stress responses were compared in tobacco plants constitutively over-expressing a gene for the Pro biosynthetic enzyme Δ(2)-pyrroline-5-carboxylate synthetase (P5CSF129A; EC 2.7.2.11/1.2.1.41) and in the corresponding wild-type. Significantly enhanced Pro production in the transformant coincided with a more negative leaf osmotic potential (both at control conditions and following stress) and enhanced production of protective xanthophyll cycle pigments. Heat stress (40 °C) caused a transient increase in the level of bioactive cytokinins, predominantly N(6)-(2-isopentenyl)adenosine, accompanied by down-regulation of the activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (EC 1.4.3.18/1.5.99.12). No significant differences were found between the tested genotypes. In parallel, a transient decrease of abscisic acid was observed. Following drought stress, bioactive cytokinin levels decreased in the whole plants, remaining relatively higher in preferentially protected upper leaves and in roots. Cytokinin suppression was less pronounced in Pro transformants. Exposure to heat stress (40 °C for 2h) at the end of 10-d drought period strongly enhanced the severity of the water stress, as indicated by a drop in leaf water potential. The activity of cytokinin oxidase/dehydrogenase was strongly stimulated in upper leaves and roots of stressed plants, coinciding with strong down-regulation of bioactive cytokinins in whole plants. Combined heat and drought stress resulted in a minor decrease in abscisic acid, but only in non-wilty upper leaves. Both stresses as well as their combination were associated with elevation of free auxin, indolylacetic acid, in lower leaves and/or in roots. Auxin increase was dependent on the stress strength. After rehydration, a marked elevation of bioactive cytokinins in leaves was observed. A greater increase in cytokinin content in Pro transformants indicated a mild elevation of their stress tolerance.

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

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.

Tomato (Solanum lycopersicum L.) SlIPT3 and SlIPT4 isopentenyltransferases mediate salt stress response in tomato

BackgroundCytokinins (CKs) are involved in response to various environmental cues, including salinity. It has been previously reported that enhancing CK contents improved salt stress tolerance in tomato. However, the underlying mechanisms of CK metabolism and signaling under salt stress conditions remain to be deciphered.ResultsTwo tomato isopentenyltransferases, SlIPT3 and SlIPT4, were characterized in tomato and Arabidopsis. Both proteins displayed isopentenyltransferase (IPT) activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. SlIPT3 and SlIPT4 were affected by the endogenous CK status, tightly connected with CKs feedback regulation, as revealed by hormonal treatements. In response to salt stress, SlIPT3 and SlIPT4 were strongly repressed in tomato roots, and differently affected in young and old leaves. SlIPT3 overexpression in tomato resulted in high accumulation of different CK metabolites, following modifications of CK biosynthesis-, signaling- and degradation-gene expression. In addition, 35S::SlIPT3 tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and K+/Na+ ratio retention. Involvement of SlIPT3 and SlIPT4 in salt stress response was also observed in Arabidopsis ipt3 knock-out complemented plants, through maintenance of CK homeostasis.ConclusionsSlIPT3 and SlIPT4 are functional IPTs encoded by differently expressed genes, distinctively taking part in the salinity response. The substantial participation of SlIPT3 in CK metabolism during salt stress has been determined in 35S::SlIPT3 tomato transformants, where enhancement of CKs accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response.

Antioxidant protection during ageing and senescence in chloroplasts of tobacco with modulated life span.

We studied changes in antioxidant protection during ageing and senescence in chloroplasts of tobacco (Nicotiana tabacum L., cv. Wisconsin) with introduced SAG12 promoter fused with ipt gene for cytokinin synthesis (transgenic plants with increased levels of cytokinins, SAG) or without it (control). Old leaves of SAG plants as well as their chloroplasts maintained higher physiological parameters compared to controls; accordingly, we concluded that their ageing was diverted due to increased cytokinin content. The chloroplast antioxidant protection did not decrease as well. Although antioxidant protection usually decreased in whole leaves of senescing control plants, ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) activity, which maintained the high redox state of ascorbate, increased in chloroplasts of old control leaves. Copyright

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 3rd 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.

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 μmol m−2 s−1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 μmol m−2 s−1 during further ex vitro growth (in the period of 7th – 17th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 μmol m−2 s−1 (3 % LL) or without sucrose either under PFD of 50 μmol m−2 s−1 or 200 μmol m−2 s−1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.

Effects of exogenous nitric oxide on photosynthesis

Nitric oxide (NO) is an important signalling molecule with diverse physiological functions in plants. In plant cell, it is synthesised in several metabolic ways either enzymatically or nonenzymatically. Due to its high reactivity, it could be also cytotoxic in dependence on concentration. Such effects could be also mediated by NO-derived compounds. However, the role of NO in photosynthetic apparatus arrangement and in photosynthetic performance is poorly understood as indicated by a number of studies in this field with often conflicting results. This review brings a short survey of the role of exogenous NO in photosynthesis under physiological and stressful conditions, particularly of its effect on parameters of chlorophyll fluorescence.