The <Emphasis Type="Italic">trans</Emphasis> and <Emphasis Type="Italic">cis</Emphasis> zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.     

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.     

Altitude variation in chilling tolerance among natural populations of <Emphasis Type="Italic">Elymus nutans</Emphasis> in the Tibetan Plateau

Low temperatures limit plant growth, development, and reproductive success. A series of complex adaptive responses in plants evolved to withstand this environmental challenge. Here, eight accessions of Elymus nutans, which originated in Tibet at altitudes between 3720 and 5012 m above sea level, were used to identify heritable adaptations to chilling stress. Dynamic responses of phytohormone, sugar, and gene expression levels related to chilling tolerance were analyzed. During the initial stage of chilling stress (0–24 h), some high-altitude E. nutans accessions exhibited rapid increases in abscisic acid (ABA), jasmonic acid (JA), and zeatin content. This coordinated with decreases in the levels of auxin (IAA), salicylic acid (SA), gibberellins (GA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). EnCBF9 and EnCBF14 expression in the high-altitude accessions, Baqing, Xainza, Damxung, and Ali, increased within 1 h of chilling exposure, while chilling induction of EnCOR14a was detected after 3 h of chilling stress. Accessions from high altitudes displayed an increased sucrose and raffinose accumulation and a reduced degradation of chlorophyll under chilling stress. After 24–120 h of chilling exposure, plant adaptation to the chilling treatment was associated with a lower accumulation of ABA and moderate rise of zeatin, IAA, GA, ACC, SA, and JA. EnCBF9, EnCBF14, and EnCOR14a genes were down-regulated during the late stage of chilling stress. Taken together, the dynamic responses of phytohormones and sugars, and the higher expression of the EnCBFs and EnCOR genes play critical roles in the acclimation to chilling in high-altitude accessions of E. nutans, thereby allowing them to achieve higher chilling tolerance.     

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 gibberellin on <Emphasis Type="Italic">Arabidopsis</Emphasis> growth,development, and hormonal balance under green and blue light

The role of gibberellins in plant morphology under selective light was studied. A comparison of the effects of green and blue light on growth, development, and hormonal balance was performed for two Arabidopsis thaliana (L.) Heynh. (ecotype Landsberg erecta lines: wild type Ler and its ga4-1 mutant with suppressed GA_4/1 synthesis. The absence of active GA_4/1 from ga4-1 mutant determined its retarded growth, slowed passing through developmental phases, suppressed apical dominance, and reduced seed productivity. The retarded growth and development of the mutant was related to changed hormonal balance in them as compared to wild-type line: IAA content and the IAA/ABA ratio were declined, zeatin riboside and ABA accumulated. Green light retarded stem elongation and branching, reduced leaf specific surface density and plant seed productivity, and retarded plant transition to reproduction to a greater degree at GA_4/1 deficit (ga4-1) than at its normal content (Ler).     

Dynamics of growth and the content of endogenous phytohormones during kidney bean scoto-and photomorphogenesis

The dynamics of growth and the contents of free and bound endogenous IAA, gibberellins (GA), cytokinins (zeatin and its riboside), and ABA in kidney bean plants (Phaseolus vulgaris L., cv. Belozernaya) grown in darkness or in the light was studied. Phytohormones were quantified in 5–15-day-old plants by the ELISA technique. Plant growth and phytohormone content were shown to depend on plant age and the conditions of illumination. During scotomorphogenesis, changes in the biomass and hypocotyl length were highly correlated with the content of GA, whereas during photomorphogeneses, these parameters were correlated with the content of zeatin. In darkness, epicotyl growth displayed a positive correlation with the content of GA, whereas in the light, the correlation was negative. Growth characteristics of the primary leaves were shown to correlate with IAA in darkness and with GA and zeatin in the light. At a low concentration of cytokinins in illuminated leaves, cell divisions occurred, whereas, at the higher cytokinin concentrations, cell expansion occurred. The highest content of GA was characteristic of leaves in the period of growth cessation. ABA accumulated during active leaf and root elongation and biomass increment in the light and during hypocotyl growth in darkness. After plant illumination, the ratio of auxins to cytokinins increased in bean roots and decreased in their epicotyls. Thus, light changed the developmental programs of bean plants, which was manifested in the changed rate and duration of growth of various organs (root, hypocotyl, epicotyl, and leaf). Some mechanisms of light action depended on the contents of IAA, ABA, GA, and cytokinins and the ratios between these phytohormones. Differences between scotonorphogenesis of mono-and dicotyledonous plants are discussed in relation to the levels of phytohormones in them.     

γ-Irradiation of Barley Seeds and Its Effect on the Phytohormonal Status of Seedlings

We investigated the effect of γ-irradiation (4–50 Gy) of barley seeds (Hordeum vulgare L., cv. Nur) on the content of endogenous phytohormones–stimulators of plant growth and development: indol-3-acetic acid (IAA), indolyl-3-butyric acid (IBA), zeatin and abscisic acid (ABA). The ratio (IAA + IBA + zeatin)/ABA from the third to the seventh day of germination has been measured. It was shown that the changes in the content of phytohormones as a function of the radiation dose were nonlinear. In the dose range of 4–20 Gy, phytohormones balance was changed due to increased content of growth stimulators and decreased ABA content. Using a dose of 50 Gy led primarily to a decrease in the content of growth stimulators and an increase in ABA content, and the ratio (IAA + IBA + zeatin)/ABA shifted toward ABA content.     

Regulation of root development in <Emphasis Type="BoldItalic">Arabidopsis thaliana</Emphasis> by phytohormone-secreting epiphytic methylobacteria

In numerous experimental studies, seedlings of the model dicot Arabidopsis thaliana have been raised on sterile mineral salt agar. However, under natural conditions, no plant has ever grown in an environment without bacteria. Here, we document that germ-free (gnotobiotic) seedlings, raised on mineral salt agar without sucrose, develop very short root hairs. In the presence of a soil extract that contains naturally occurring microbes, root hair elongation is promoted; this effect can be mimicked by the addition of methylobacteria to germ-free seedlings. Using five different bacterial species (Methylobacterium mesophilicum, Methylobacterium extorquens, Methylobacterium oryzae, Methylobacterium podarium, and Methylobacterium radiotolerans), we show that, over 9 days of seedling development in a light-dark cycle, root development (hair elongation, length of the primary root, branching patterns) is regulated by these epiphytic microbes that occur in the rhizosphere of field-grown plants. In a sterile liquid culture test system, auxin (IAA) inhibited root growth with little effect on hair elongation and significantly stimulated hypocotyl enlargement. Cytokinins (trans-zeatin, kinetin) and ethylene (application of the precursor ACC) likewise exerted an inhibitory effect on root growth but, in contrast to IAA, drastically stimulated root hair elongation. Methylobacteria are phytosymbionts that produce/secrete cytokinins. We conclude that, under real-world conditions (soil), the provision of these phytohormones by methylobacteria (and other epiphytic microbes) regulates root development during seedling establishment.     

Effect of the nitrogen-containing salt on the content of cytokinins in detached wheat leaves

The dynamics of the cytokinin content in detached leaves of wheat (Triticum durum, cv. Bezenchukskaya 139) seedlings moistened with ammonium nitrate or water (control) was studied by immunoenzyme analysis. Leaf treatment with water was accompanied by a transient accumulation of cytokinins, maybe due to their release from their O-glucosylated forms. An increase in the contents of zeatin and its riboside after their initial decrease in detached leaves treated with ammonium nitrate could not occur due to their release from stored forms (nucleotides or O-glucosides) because the contents of zeatin and its riboside increased simultaneously with the content of stored cytokinins. The accumulation of isopentenyladenosine and zeatin nucleotide, which occurred simultaneously with an increase in the content of zeatin and zeatin riboside, permits a supposition that cytokinins can be synthesized in detached wheat leaves treated with ammonium nitrate.     

Hormone-Induced Gene Expression During Gravicurvature of <Emphasis Type="Italic">Brassica</Emphasis> Roots

To investigate the spatial and temporal dependence of hormonal regulation during gravitropism, we compared the effects of root cap application of indole-3-acetic acid (IAA) and abscisic acid (ABA) with gene expression changes occurring naturally during gravitropic reaction of Brassica rapa roots. The expression of auxin, ABA, and metabolism-related genes in the tip, elongation zone, and maturation zone varied with time, location, and hormone concentration and characterized polar auxin transport. IAA was transported readily shootward and inhibited growth more than ABA. Expression of PIN3 and IAA5 in the elongation zone showed downregulation on the convex but upregulation on the concave side. Both PIN7 and IAA5 responded near maximally to 10^?8 M IAA within 30 min, suggesting that auxin activates its own transport system. Ubiquitin 1 (UBQ1) responded after a lag time of more than 1 h to IAA. The metabolic control gene Phosphoenolpyruvate carboxylase 1 (PEPC1) was more sensitive to ABA but upregulated by high concentrations of either hormone. The time course and duration of gene activation suggests that ABA is not involved in gravitropic curvature, differential elongation is not simply explained by IAA-induced upregulation, and that reference genes are sensitive to auxin.     

Dependence of root biomass accumulation on the content and metabolism of cytokinins in ethylene-insensitive plants

Diverse functions of ethylene in plants may depend on its ability to interact with other hormones. We studied the participation of ethylene in the regulation of accumulation and metabolism of cytokinins comparing ethylene-insensitive mutant plants of arabidopsis (Arabidopsis thaliana [L.] Heynh., etr1-1) with the plants of original ecotype Columbia (Col-0). Because cytokinins can regulate growth of both leaves and roots, we determined the weights of these organs and the ratio between them. The content of zeatin and its riboside in the roots of etr1-1 plants was two times greater than in Col-0 plants, which could be accounted for by inhibition of conversion of these forms of cytokinins into 9-N-glucosides. In the leaves of mutant plants, expression of IPT3 gene responsible for the synthesis of cytokinins was more intense than in Col-0 plants, which could also contribute to a rise in the content of cytokinins. In this case, the weight of roots in etr1-1 mutants was lower than in the plants of original ecotype. Because high concentrations of cytokinins can inhibit root growth, suppression of accumulation of their biomass in mutant plants may be related to a greater content of cytokinins therein. The obtained results suggest that ethylene can suppress accumulation of cytokinins and, thereby, maintain redistribution of biomass in favor of the roots, which is important for plant adaptation to a shortage of water and ions.     

Effect of different benzyladenine time pulses on the endogenous levels of cytokinins,indole-3-acetic acid and abscisic acid in micropropagated explants of Actinidia deliciosa

Actinidia deliciosa apical shoots were cultured in MS liquid medium with different benzyladenine (BA) pulses and using cellulose plugs as support for the explants. Abscisic acid (ABA), indole-3-acetic acid (IAA) and some cytokinins (Cks) [zeatin (Z), dihydrozeatin (DHZ), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), N⁶-isopentenyladenine (iP) and N⁶-isopentenyladenosine (iPR)] levels were measured in leaves from explants cultured both in the absence and in the presence of BA (4.4 μM) for 30 min, 1, 2 and 35 d. Analyses were carried out after three subcultures at the end of the multiplication phase and after 31 d in ex vitro conditions. A clear relationship between the endogenous content of phytohormones and the growth characteristic of kiwifruit microplants could be confirmed. Microplants with the best growth characteristic were those cultured in presence of BA during 1 d. They were characterised by higher contents of IAA than the others studied, as well as by higher values in the IAA/Cks and IAA/ABA ratios, both at the end of the multiplication phase and after the acclimatisation period. Taken all together, these ratios at the end of the multiplication phase could be used as growth indicators of A. deliciosa explants behaviour under ex vitro conditions.     

Effect of <Emphasis Type="Italic">Rol</Emphasis> Transgenes,IAA, and Kinetin on Starch Content and the Size of Starch Granules in Tubers of <Emphasis Type="Italic">In Vitro</Emphasis> Potato Plants

Stem cuttings were produced from Solanum tuberosum L., cv. Desiree, plants and their transgenic forms harboring rolB and rolC genes from Agrobacterium rhizogenes. Plants were cultured on hormone-free Murashige and Skoog nutrient medium (MS) and on MS supplemented with IAA or kinetin. In microtubers developed on these cuttings, we estimated the content of starch and the number and size of starch granules. Expression of rol genes changed these indices: in tubers of rolC transformants, a greater number of small granules were produced, whereas in tubers of rolB transformants, a fewer number of large granules were developed as compared with wild-type plants. Expression of rol genes did not affect starch content during the first three weeks of cutting culturing but increased it by 15–30% in five-week-old tubers. IAA addition to MS medium increased starch content and the size of starch granules in control plants and rolB tubers by 10–30%, whereas kinetin did not exert any significant influence. The effects of rol transgenes on the initiation and termination of starch granule development are discussed.     

Growth characteristics and endosperm structure of superior and inferior spikelets of indica rice under high-temperature stress

Heat stress severely reduces rice yield and quality; however, differences between the superior, early-flowering and inferior, later-flowering spikelets of indica rice in response to high-temperature stress during grain filling remain unclear. This study investigated the effects of high temperature (HT, 33.6/20.7 °C day/night) on growth, endosperm structure, and hormone and polyamine content of superior and inferior spikelets of heat-sensitive (SG-1) and heat-tolerant (HHZ) indica cultivars. The HT decreased fertilization rate, caused earlier grain filling, and reduced duration of grain filling, thus resulting in decreased grain mass and a poor endosperm structure. In addition, soluble sugar and sucrose content increased, and starch synthesis decreased by HT at the early stage of grain filling. The HT increased polyamine [spermidine (Spd) and spermine (Spm)] and abscisic acid (ABA) content, but reduced zeatin (Z) + zeatin riboside (ZR) and indole-3-acetic acid (IAA) content in the grains. Such effects were more apparent in the inferior than superior spikelets; however, the inferior spikelets of SG-1 were more affected than those of HHZ. At the middle grain filling stage, HT produced little difference between the two cultivars. Our results suggest that the poor development of inferior spikelets of SG-1 under the HT could be attributed, at least in part, to the changed content and ratios of free polyamines [putrescine (Put), Spd, and Spm] and phytohormones (Z+ZR, IAA, and ABA) and the conversion efficiency of sucrose into starch.     

Dynamic changes in carbohydrate metabolism and endogenous hormones during <Emphasis Type="Italic">Tulipa edulis</Emphasis> stolon development into a new bulb

The stolon is the main asexual reproductive organ of Tulipa edulis (Miq.) Baker. It has a special morphology and can develop into a new bulb for propagation. In the current greenhouse experiment, the dynamic changes in carbohydrates and related enzymes, protein and endogenous hormones during T. edulis stolon development were investigated. The results showed that soluble sugar levels were basically declining, whereas starch and protein content rose continuously during stolon development. The adenosine diphosphoglucose pyrophosphorylase (AGPase) activity peaked in the initial swelling stage and stayed a relative high level in the middle swelling stage; sucrose synthase (SS), soluble starch synthase (SSS) and granule-bound starch synthase (GBSS) activities followed the same law that showed rising trends during stolon development. SS activity was significantly inversely related to sucrose content but had significantly positive relations with starch content, SSS and GBSS activities. Gibberellin (GA), indole-3-acetic acid (IAA) and zeatin riboside (ZR) peaked in the initial swelling stage and maintained high levels in the middle swelling stage; they then decreased significantly in the later swelling stage. A substantial increase was observed in abscisic acid (ABA) content until the middle swelling stage, followed by a significant reduction in the later swelling stage. The ratios of ABA to IAA, GA and ZR reached their lowest levels in the initial swelling stage. In conclusion, T. edulis stolon development is a process of new bulb morphogenesis along with the starch accumulation catalyzed by AGPase, SSS and GBSS, using the product of sucrose cleavage caused by SS. Initial low ABA content and low ratios of ABA to IAA, GA and ZR, together with the GA, IAA and ZR of high-content, soluble sugars worked more efficiently to induce new bulb formation.     

Comparative analysis of the effects of chitosan and common plant growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Ipomoea purpurea</Emphasis> (L.) Roth from nodal explants

This study aimed to evaluate the effects of partially N-acetylated chitosans with a degree of acetylation (DA) of 10% on in vitro propagation of an ornamental plant, Ipomoea purpurea, by emphasizing the importance of the degree of polymerization (DP) on in vitro plant development. The effects of either a chitosan oligomer mixture with a DP between 2 and 15 (5.0, 10.0, and 20.0 mg L^?1) or chitosan polymer with a DP of 70 were compared with commonly used cytokinins [6-benzylaminopurine (BAP) and kinetin (KIN) at 0.5, 1.0, 2.0, and 4.0 mg L^?1] and auxins [indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) at 0.25, 0.5, 1.0, and 2.0 mg L^?1]. The nodal explants used in this study were taken from donor plants obtained by germination of the seeds. The results indicated that all chitosan treatments had positive effects on the shoot induction, but only the oligomer mixture at 5 mg L^?1 gave the best results for mean shoot number, shoot length, and leaf number, compared to the other treatments and control. Also, all chitosan treatments increased mean number of roots and triggered adventitious root induction. However, root elongation was decreased in the presence of chitosan in the medium. The root elongation-inhibitory effects of chitosan become clearer in the presence of oligomer mixture. In general, chitosan had similar effects with tested cytokinins rather than auxins. The results of this study suggested that the application of chitosan oligomers—rather than polymers—can be an eco-friendly and effective alternative to synthetic cytokinins in horticulture.     

Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.