Cytokinin Activity in Stolons and Tubers of Solanum tuberosum during the Period of Tuberization

In water-culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), changes in cytokinin activity in the stolon tips and newly formed tubers during tuberization were studied. Tuberization was induced by withdrawing nitrogen from the nutrient solution. — The cytokinin activity was low in the stolon tips prior to tuberization, but increased considerably in both stolon tips and young tubers during tuberization. At the same time qualitative changes in the cytokinin spectrum occurred. These qualitative changes are reversible if ‘regrowth’ of young tubers is brought about by a sudden high supply of nitrogen. — Despite the close correlation between tuberization and cytokinin activity, it is assumed that cytokinins are not directly responsible for the onset of tuberization, although they play an important role in tuber growth.     

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Expressed in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors, and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction with isoprenoid cytokinins (N ⁶ -(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol (DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q₀). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference for cytokinin glycosides, especially N ⁶ -(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles.     

Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn.

Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants.     

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Molecular Cloning and Characterization of a Cytokinin Dehydrogenase Gene from Upland Cotton (Gossypium hirsutum L.)

Cytokinins are plant hormones that play crucial roles in plant growth and development. Cytokinin dehydrogenase (CKX), regarded as a main negative regulator in cytokinin metabolism in plants, irreversibly degrades cytokinins into adenine/adenosine moiety. A CKX homologous gene, designated GhCKX, was cloned from upland cotton (Gossypium hirsutum L.). Transgenic tobacco plants over-expressing GhCKX showed a typical cytokinin-deficient phenotype, while CKX-silenced tobacco plants exhibited cytokinin over-producing phenotype. Tissue specifically enhancing the expression of GhCKX in the ovule epidermis of transgenic cotton led to a significant decrease of trans-zeatin and trans-zeatin riboside contents in the ovule. The decline of cytokinins resulted in a significant decrease in fiber initials on a single ovule. Our results indicate that GhCKX encodes a functional CKX, and cytokinins may be required for the initiation of cotton fiber cells.     

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties,activity, and expression during tuber dormancy progression

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [³H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.     

Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics

GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA₁ and GA₄. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato.     

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.     

Effect of ethylene on the endogenous cytokinin and gibberellin levels in tuberizing potatoes

High concentrations of 2-chloroethylphosphonic acid inhibited tuberization on aged potato tubers (Solanum tuberosum) that had been predisposed to the little tuber disorder. As a result of this treatment sprouts developed which contained relatively high levels of endogenous gibberellins and which elongated normally. The endogenous cytokinin levels in the different treatments did not change appreciably. It is suggested that tuberization is prevented by ethylene either as a direct inhibition of cell division or that it prevents the endogenous cytokinins from functioning. Irrespective of the mode of action of ethylene, cell division apparently is the primary process affected, the result being that storage tissue required for the accumulation of starch is not formed.     

Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: Heterologous expression,purification and properties

The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N⁶-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q₀). In slightly acidic conditions (pH 5.0), N⁶-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N⁶-(2-isopentenyl)adenine 9-riboside-5′-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N⁶-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.     

Relation between Nitrogen Nutrition, Cytokinin Activity and Tuberization in Solanum tuberosum

In water-culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), the influence of tuberization initiated by a 7-day period of nitrogen withdrawal (discont. N) on the cytokinin activity in shoots, roots and exudate was studied. Plants with a constant supply of nitrogen (cont. N) were used as control. — Whereas no tuberization could be observed with cont. N, discont. N led to tuberization already 2 days after nitrogen withdrawal and all plants had been induced after another 4 days. In the roots of plants with discont. N, there was a temporary increase in cytokinin activity, whereas the activity decreased steadily with cont. N. In the exudate, cytokinin activity was greatly reduced during nitrogen withdrawal, whereas this activity in the exudate increased steadily with cont. N. — In the shoot with cont. N cytokinin activity decreased steadily, but with discont. N, after an initial decrease, the activity increased steeply. This increase is mainly or exclusively caused by a shift between the water-soluble and butanol-soluble fractions of the cytokinins in favour of the latter. The shift in cytokinin activity of the shoot is assumed to be in causal connection with an increased photosynthetic activity after the onset of tuber growth as ‘sink’ for assimilates.     

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−³H]trans-zeatin ([2−³H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−³H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−³H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.     

Photosynthesis and protective mechanisms during ageing in transgenic tobacco leaves with over-expressed cytokinin oxidase/dehydrogenase and thus lowered cytokinin content

The content of cytokinins (CKs), the plant inhibitors of the final phase of plant development, senescence, is effectively controlled by irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). In transgenic tobacco, denoted as AtCKX, with over-expressed CKX causing lowered CK content, we investigated changes in the time courses of chlorophyll (Chl) and xanthophyll (violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein) contents. We also determined parameters of slow Chl fluorescence kinetics such as minimum Chl fluorescence yield in the darkadapted state F₀, maximum quantum yield of PS2 photochemistry (F_v/F_m), maximum ratio of quantum yields of photochemical and concurrent non-photochemical processes in photosystem 2 (PS2), F_v/F₀, non-photochemical quenching (NPQ), and effective quantum yield of photochemical energy conversion in PS2 (Φ₂). We used three different developmental leaf stages, old, mature, and young, and compared this with time courses of these characteristics in leaves with natural CK levels. The parameters F_v/F_m, F_v/F₀, and Φ₂ were unchanged during ageing in AtCKX plants in contrast to control ones where a significant decrease in old leaves was found. In control plants F₀ increased during ageing, but in the oldest leaf a considerable decrease was observed. This could indicate progressive damage to PS2 reaction centres and then detachment and rapid degradation of Chl. This is in agreement with time course of Chl content. NPQ decreased with age and was similar in both plant types. We observed a decline of xanthophyll contents in the oldest leaves in both plant types, but the contents were enhanced in AtCKX compared to control plants, especially of neoxanthin. The higher xanthophyll contents in the transgenic plants contribute to a better photoprotection and the fluorescence parameters indicated that photosynthetic apparatus was in better condition compared to control and it consequently postponed the onset of leaf senescence.     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.     

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the <Emphasis Type="Italic">PHYB</Emphasis> Gene from <Emphasis Type="Italic">Arabidopsis</Emphasis>

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.     

The effect of an elevated cytokinin level using the ipt gene and N 6-benzyladenine on single node and intact potato plant tuberization in vitro

Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L^-1 N ⁶-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.Abbreviations BA N ⁶-benzyladenine - PCR polymerase chain reaction - HPLC high performance liquid chromatography - ELISA enzyme-linked immunosorbent assay - NAA -naphthylacetic acid