Low Cytokinin Concentrations Research Articles

Seed dormancy mechanism and dormancy-breaking methods in wild raspberry (Rubus fraxinifolius Poir.)

Raspberries are subtropical plants that contain high levels of vitamin C, antibacterial and anti-inflammatory. They can potentially be developed as horticultural and medicinal plants. Dormancy is a challenge in the cultivation of raspberries (Rubus fraxinifolius Poir.). This study was conducted as two separate experiments. The first experiment aimed to identify the dormancy mechanism of R. fraxinifolius seed. In a two-factor factorial design, the first factor was seed storage, as unstored and three-month-stored, and the second factor was chemical-immersed treatment consisting of control, H2SO4, acetone, GA3, KNO3, H2SO4-GA3, acetone-GA3, H2SO4-KNO3, acetone-KNO3. The second experiment was aimed at determining dormancy-breaking methods for R. fraxinifolius seeds. In main plots were filter paper and cocopeat germination substrates. The subplots included control, immersed with distilled water, H2SO4, ultrafine bubble water, and temperature treatment at −80 °C, 50 °C, and 70 °C. The germination of unstored and three-month-stored seeds increased after H2SO4 treatment (36 to 82% and 82 to 94%, respectively). Seed germination increased after three months of storage. There was an increase in cytokinin hormone levels along with germination enhancement. The seeds went into physical dormancy because their seed coat was hard, and they went into physiological dormancy because of low cytokinin concentration. Stratification at 50 °C increased germination (78.5 to 93.0%), reduced dormancy intensity (15 to 6.5%), and increased the percentage of the speed of germination (1.99 to 3.12 ) on filter paper substrate.

Asymbiotic germination of Prasophyllum (Orchidaceae) requires low mineral concentration

Context Asymbiotic germination is used for propagation of orchids and to assess seed viability. However, asymbiotic germination media remain undeveloped for Prasophyllum (Orchidaceae), a genus with 39 threatened species in southern Australia. Aims To develop asymbiotic germination and replate media for Prasophyllum, and determine whether survival of asymbiotically grown seedlings under nursery conditions is enhanced through the addition of a mycorrhizal fungus to potting media. Methods Using Prasophyllum frenchii as a model, we tested 81 asymbiotic germination medium combinations. The three best-performing media were then compared against four untested commercial media, with and without added coconut water and/or banana. The best-performing medium was trialled on 18 additional Prasophyllum species. Twenty-four replate media were then tested for their ability to support seedling development. We measured the survival of seedlings under nursery conditions with and without the addition of mycorrhizal Ceratobasidium fungus to potting media. Key results Low concentrations of minerals, auxins and cytokinins were necessary for asymbiotic germination of P. frenchii, although germination was highest overall on the commercial medium W9.5 with 5% coconut water. The most effective replate medium was W9 with 5% banana. However, survival of seedlings was 1.7%, 2 years after transferral to potting media in the nursery, regardless of the addition or absence of a mycorrhizal fungus. Conclusions Asymbiotic media can effectively germinate Prasophyllum seed, although deflasked seedlings have low survival rates. Implications Our media are broadly applicable to assess seed viability in Prasophyllum. However, poor survival of asymbiotically grown seedlings contrasts with recently published evidence of high seedling survival using symbiotic propagation methods, indicating that plants should be propagated symbiotically for conservation programs.

Involvement of cytokinins in STOP1-mediated resistance to proton toxicity

STOP1 (sensitive to proton rhizotoxicity1) is a master transcription factor that governs the expression of a set of regulatory and structural genes involved in resistance to aluminum and low pH (i.e., proton) stresses in Arabidopsis. However, the mechanisms and regulatory networks underlying STOP1-mediated resistance to proton stresses are largely unclear. Here, we report that low-pH stresses severely inhibited root growth of the stop1 plants by suppressing root meristem activities. Interestingly, the stop1 plants were less sensitive to exogenous cytokinins at normal and low pHs than the wild type. Significantly, low concentrations of cytokinins promoted root growth of the stop1 mutant under low-pH stresses. Moreover, lateral and adventitious root formation was stimulated in stop1 and by low-pH stresses but suppressed by cytokinins. Further studies of the expression patterns of a cytokinin signaling reporter suggest that both the loss-of-function mutation of STOP1 and low-pH stresses suppressed cytokinin signaling outputs in the root. Furthermore, the expression of critical genes involved in cytokinin biosynthesis, biodegradation, and signaling is altered in the stop1 mutant in response to low-pH stresses. In conclusion, our results reveal a complex network of resistance to low-pH stresses, which involves coordinated actions of STOP1, cytokinins, and an additional low-pH-resistant mechanism for controlling root meristem activities and root growth upon proton stresses.

Obtaining of the fertile sunflower-regenerants (Helianthus annuus L.) by in vitro organosesis

Sunflower (Helianthus annuus L.) is one of the mainoilseeds in the world and by far one of the hardestcrops to cultivate in vitro. Currently, there is no registrated transgenic sunflower (Helianthus annuus L.) variety (clone, line, F1 hybrid, population). One ofthe main problems for obtaining transgenic plants isan efficient in vitro regeneration system, which willmake it possible to obtain morphologically typical fertile regenerants. It is known that the regenerative ability of sunflower can be studied in two ways: somatic embryogenesis and direct organogenesis. Due to the different ratio of growth regulators such as auxin and cytokinins, it is possible to influence the frequency of regeneration from different types of tissues. Thus, at high concentrations of auxins and low concentrations of cytokinins, it is possible to induce root regeneration. And for the induction of shoot regeneration, it is better to use high concentrations of cytokinin and lowconcentrations of auxins. However, knowing differentapproaches and methods of sunflower cultivation invitro regeneration culture, today there is no universalprotocol that is suitable for all sunflower genotypes.That is why the aim of our work was to investigatethe regenerative capacity of sunflower lines and develop an effective system that will later be used forgenetic research in order to improve economicallyvaluable traits of sunflower. This article presents data on sunflower regeneration (Helianthus annuus L.) by direct organogenesis.Cotyledons of immature seeds (21 days after pollination) were used as explants. The induction andproliferation of adventive buds was tested in a basicmedium with different concentrations of growth regulators. It was found that the optimal conditions for the induction and proliferation of adventive buds is a modified MS medium supplemented with vitamins for Gamborg, 5 mg/l AgNO3, 2 mg/l 2–isopentenyladenine (2–iP), 0.5 mg/l indole–3–acetic acid (IAA), 0.1 mg/L thidiazuron (TDZ).The elongation of adventitious shoots was carried out on a modified MS medium with Gamborg vitamins, 5 mg/L AgNO3, 1 mg/L 2-IP, 0.5 mg/L N6-benzylaminopurine (BAP). We were the development of an effective in vitro rooting system allowed to obtain seeds.

Transcriptomic differences between male and female Trachycarpus fortunei

Trachycarpus fortunei (Hook.) is a typical dioecious plant, which has important economic value. There is currently no sex identification method for the early stages of T. fortunei growth. The aim of this study was to obtain expression and site differences between male and female T. fortunei transcriptomes. Using the Illumina sequencing platform, the transcriptomes of T. fortunei male and female plants were sequenced. By analyzing transcriptomic differences, the chromosomal helical binding protein (CHD1), serine/threonine protein kinase (STPK), cytochrome P450 716B1, and UPF0136 were found to be specifically expressed in T. fortunei males. After single nucleotide polymorphism (SNP) detection, a total of 12 male specific sites were found and the THUMP domain protein homologs were found to be male-biased expressed. Cytokinin dehydrogenase 6 (CKX6) was upregulated in male flowers and the lower concentrations of cytokinin (CTK) may be more conducive to male flower development. During new leaf growth, flavonoid and flavonol biosynthesis were initiated. Additionally, the flavonoids, 3′,5′-hydroxylase (F3′5′H), flavonoids 3′-hydroxylase, were upregulated, which may cause the pale yellow phenotype. Based on these data, it can be concluded that inter-sex differentially expressed genes (DEGs) and specific SNP loci may be associated with sex determination in T. fortunei.

Positive impact of vermicompost leachate on salt stress resistance in tomato (Solanum lycopersicum L.) at the seedling stage: a phytohormonal approach

Vermicompost leachate (VCL) produced by earthworms is a valuable biostimulant but its hormonal impact on salt-treated plants remains elusive. Plants of Solanum lycopersicum L. were cultivated in nutrient solution and exposed during 7 days to 125 mM NaCl in the presence or absence of VCL (18 mL.L−1). Mineral nutrition, water and hormonal status were quantified in roots, young and old leaves and analyzed in relation to phytohormone content of VCL. VCL improved plant growth and reduced Na+ accumulation in salt-stressed plants. It delayed senescence in young leaves through a decrease in ethylene synthesis and an increase in proline and anthocyanin contents. VCL contains high amounts of salicylic acid, benzoic acid and aminocyclopropane carboxylic acid (ACC) but low concentrations of jasmonates, cytokinins and proline. VCL did not increase abscisic acid content in salt-stressed plants and did not lead to ACC accumulation while it increased jasmonate accumulation and modified the pattern of cytokinin profile with an increase in dihydrozeatin-types in old leaves and N6-(∆2-isopentenyl)adenine-types in young ones. VCL reduces the impact of salinity on leaf senescence, which is related to its impact on endogenous phytohormones rather than to a passive absorption of exogenous hormonal compounds.

Bazı siklamen türlerinde farklı doku kültürü ortamlarının gynogenesis üzerine etkileri

Cyclamen breeding is difficult due to selfing depression different ploidy levels, self-incompatibility and abortive embryo formation. Therefore, in vitro haploidisation is commonly used in cyclamen breeding. In this study, we investigated effects of different tissue culture media and plant hormones as a first of in vitro haploidisation and propagation. We used ovule explants from Cyclamen persicum and Cyclamen pseudibericum on ½MS and B5 culture mediums supplemented with different concentration of 2,4-dichlorophenoxyaceticacid (2,4-D) and 6-(γ,γ –dimethylallylamino) purine (2iP). According to results best callus formation rate was 70% obtained from B5 medium supplemented with 1.5 mg L-1 2,4-D and 0.5 mg L-1 2iP in Cyclamen persicum. It was 48% on B5 medium supplemented with 1 mg L-1 2,4-D and 0.3 mg L-1 2iP in Cyclamen pseudibericum. In Cyclamen persicum, highest embryo induction rate was 55% obtained from callus regenerated on ½MS medium supplemented with 1.5 mg L-1 2,4-D and 0.8 mg L-1 2iP. However, embryo induction was not obtained on any medium for Cyclamen pseudibericum. Overall, ½MS culture medium supplemented with high auxin and low cytokinin concentration is better for embryo induction although B5 medium was better than ½MS medium for callus formation and 42 plantlets obtained only from callus regenerated on ½MS medium supplemented with 2.0 mg L-1 2,4-D and 0.5 mg L-1 2iP.

THE EFFECT OF GROWTH REGULATORS ON THE CLONAL MICROPROPAGATION OF ACTINIDIA GENUS

Actinidia Kolomicta is the most common and frost-resistant fruiting vine. Its fruit contains a record amount of ascorbic acid and by this feature it is superior to other Actinidia species and crops like raspberries, lemon, and blackcurrant. The influence of growth regulators on the clonal micropropagation in the Actinidia genus has been studied in vitro. Two female varieties of Actinidia Kolomikta (Excellent & Moma) and one male form Actinidia Giraldi were the objects of investigation. Microcuttings with two buds were used as explants. We tested 15 variants of Murashige-Skoog (MS) medium with different ratio of growth regulators BAP, TDZ, 2-ip, IBA, NAA, L-glytamen. Activation of meristems development was the main method of clonal micropropagation. This method provides the minimum frequencies of changed forms. The best result was observed on medium supplemented with low concentrations of cytokinins: 2–3µM BAP and 4 µM 2-ip which promoted micropropagation. Combination of auxin with cytokinins has reduced the coefficient of micropropagation. The only for Moma variety on the medium containing 2,5 μM BAP +1,0 μM IBA was higher (2,5 ± 0,8). We registered no significant relationship between the used type of cytokinin (BAP, 2-ip, or TDZ) and its concentration. High concentrations of BAP (10 μM) decreased Actinidia multiplication factor from 2,5 to 1,2 and increased callus formation. Use of TDZ was not effective nevertheless. We did not identify the significant statistics coefficients of multiplication, shoot length, and number of leaves between species genotypes.

Improved Efficiency of Microspore Culture of <i>Brassica campestris</i> Ssp. <i>pekinensis</i> (Chinese Cabbage)

[Objective](1) to investigate the factors that influence microspore embryogenesis and plantlet regeneration;(2) to discuss some protocols for the induction culture of microspore-derived embryos and for rooting and transplantation of microspore-derived plantlets. [Proposed Methods] B. campestris ssp. pekinensis ‘Futian 50’ and B. campestris ssp. pekinensis ‘Changkuai’ were used as the experimental materials, then their microspores were cultured in NLN media. [Results] NAA and 2,4-D inhibits the formation of microspore-derived embryos. Low concentrations of cytokinins facilitate embryogenesis, while high concentrations inhibit embryogenesis.The combined effects of auxin and cytokinin are synergistic. However, AC inhibits embryo development. [Conclusion] The better the development of the embryos, the higher is the plantlet regeneration rate. The plant regeneration rate increased significantly on the MS culture medium supplemented with 200 mg·l-1 AC. MS medium containing 0.1 mg·l-1 NAA is the optimal medium for rooting of microspore-derived plantlets.

English

We selected two cultivars of Brassica campestris ssp. pekinensis (Chinese cabbage), ‘Futian 50’ and ‘Changkuai,’ to investigate the factors that influence microspore embryogenesis and plantlet regeneration. We have also discussed some protocols for the induction culture of microspore-derived embryos and for rooting and transplantation of microspore-derived plantlets. We obtained the following findings in our study. Although NAA promotes the development of embryo and reduces the percentage of abnormal embryos, it inhibits the formation of microspore-derived embryos. 2,4-D also inhibits the formation of microspore-derived embryos. Low concentrations of cytokinins facilitate embryogenesis, while high concentrations inhibit embryogenesis; BA has a stronger influence than zeatin (Z) on embryo induction. The combined effects of auxin and cytokinin are synergistic. Organic compounds increase the rate of formation of microspore-derived embryos. However, activated charcoal (AC) inhibits embryo development. The better the development of the embryos, the higher is the plantlet regeneration rate. The plant regeneration rate increased significantly on the MS culture medium supplemented with 200 mg·L-1 AC. The MS medium s suitable for the subculture of the regenerated plantlets. MS medium containing 0.1 mg·L-1 NAA is the optimal medium for rooting of microspore-derived plantlets.   Key words: Chinese cabbage, microspore culture, embryogenesis, plant regeneration.

Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.

Influence of root-bed size on the response of tobacco to elevated CO2 as mediated by cytokinins.

The extent of growth stimulation of C3 plants by elevated CO2 is modulated by environmental factors. Under optimized environmental conditions (high light, continuous water and nutrient supply, and others), we analysed the effect of an elevated CO2 atmosphere (700 ppm, EC) and the importance of root-bed size on the growth of tobacco. Biomass production was consistently higher under EC. However, the stimulation was overridden by root-bed volumes that restricted root growth. Maximum growth and biomass production were obtained at a root bed of 15 L at ambient and elevated CO2 concentrations. Starting with seed germination, the plants were strictly maintained under ambient or elevated CO2 until flowering. Thus, the well-known acclimation effect of growth to enhanced CO2 did not occur. The relative growth rates of EC plants exceeded those of ambient-CO2 plants only during the initial phases of germination and seedling establishment. This was sufficient for a persistently higher absolute biomass production by EC plants in non-limiting root-bed volumes. Both the size of the root bed and the CO2 concentration influenced the quantitative cytokinin patterns, particularly in the meristematic tissues of shoots, but to a smaller extent in stems, leaves and roots. In spite of the generally low cytokinin concentrations in roots, the amounts of cytokinins moving from the root to the shoot were substantially higher in high-CO2 plants. Because the cytokinin patterns of the (xylem) fluid in the stems did not match those of the shoot meristems, it is assumed that cytokinins as long-distance signals from the roots stimulate meristematic activity in the shoot apex and the sink leaves. Subsequently, the meristems are able to synthesize those phytohormones that are required for the cell cycle. Root-borne cytokinins entering the shoot appear to be one of the major control points for the integration of various environmental cues into one signal for optimized growth.

Optimization of multiple shoot induction and plant regeneration in Indian barley (Hordeum vulgare) cultivars using mature embryos

Barley is the fourth most important crop in the world. Development of a regeneration system using immature embryos is both time consuming and laborious. The present study was initiated with a view to develop a regeneration system in six genotypes of Indian barley (Hordeum vulgare) cultivars as a prerequisite to transformation. The mature embryos were excised from seeds and cultured on MS medium supplemented with high and low concentrations of cytokinins and auxins respectively. The MS medium containing 3mg/L N6-benzylaminopurine (BA) and 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) was found to be the most effective for multiple shoot formation in HOR7231 cultivar that could produce 12 shoots per explant. The other cultivars HOR4409 and HOR3844 produced a minimum number of adventitious shoots (1.33 and 1.67 respectively) on MS medium supplemented with 1mg/L BA and 0.3mg/L 2,4-D. The elongated shoots were separated and successfully rooted on MS medium containing 1mg/L indole-3-acetic acid (IAA). The response of different barley cultivars was found to be varying with respect to multiple shoot production. This is the first report of multiple shoot induction and plantlet regeneration in Indian cultivar of barley which would be useful for genetic transformation.

Pertumbuhan Planlet Nenas (Ananas comosusL. Merr.) Varietas Smooth Cayenne Hasil Kultur In Vitro pada Beberapa Konsentrasi BAP dan Umur Plantlet

<p style="text-align: justify;">The aim of this research is to study the effects of synthetic cytokinin (6-benzylaminopurine/BAP) treatments on two groups of pineapple (Ananas comosusL.Merr.) plantlets differentiated by plantlet ages. The research used the Factorial Experiment on Randomized Complete Block Design with two factors, which are BAP concentrations (0 ppm, 25 ppm, 50 ppm, and 75 ppm) and plantlet division by age grouping (52 days and 69 days), with three replications. It was then followed by Tukey’s Honestly Significant Difference Test at error level of 5%. The results show that BAP treatments with the concentration used in this research significantly inhibits the growth of pineapple plantlets observed on four variables (number of leaves, leave length, plantlet height, and plantlet diameter), while age grouping treatments didn’t show any significant effects, except on the plantlet height at 8 and 14 weeks after treatment. There are also treatment-related interactions which significantly affects the number of leaves and plantlet height. It was then suggested that lower concentrations of cytokinin are required if similar research is to be conducted in the future.</p><p>Keywords : pineapple, post-acclimatization, propagation, 6-benzylaminopurine</p>

Nitrogen and phosphorus interaction and cytokinin: Responses of the primary root of Arabidopsis thaliana and the pdr1 mutant

Nitrogen (N) and phosphorus (P) are the two most limiting nutrients for plant yield. Plants modify their metabolism and growth to cope with resources availability, consequently the integration of diverse signals is required. There is mounting evidence of N and P interaction, however, the sharing components of their signaling pathways have not been revealed yet. The pdr1 mutant has proved potentially useful in understanding the responses to nitrate (Ni), P and cytokinin. The mutation conferred pdr1 reduced root length in response to Ni under P deficiency and no effect of low cytokinin concentration. High N availability and high cytokinin caused strong root growth inhibition by different paths in wild type. Cytokinin repressed cell division, exhausted the quiescent center, caused changes in the pattern of AtPT1 expression and reduced AtACP5 expression. On the contrary, high N induced cell division as well as increased the expression of AtPT1 and AtACP5 even at high P availability. The data indicated interaction in the root modulation by N and P; and PDR1 is probably a signaling component of the nutritional status in Arabidopsis thaliana that modulates the response to N and P only partially mediated by cytokinin.

Improving Effect of Salicylic Acid on the Multipurpose Tree <i>Ziziphus spina-christi</i> (L.) Willd Tissue Culture

Salicylic acid (SA) is one of numerous phenolic compounds found in plants with numerous important physiological events. Exogenously application of Salicylic acid (SA) is useful for the growth and development of plants. This investigation aimed to study the improving effect of salicylic acid on the tissue culture of Ziziphus spina-christi. To study its improving effect were used shoot-tip explants cultured on Murashige and Skoog (MS) solid medium supplemented with different concentrations and various combinations of auxins, cytokinins and Salicylic acid. Media without growth regulators enhanced the growth and elongation of shoot-tip explants not its proliferation. Lower concentrations of cytokinin were better for shoot-tip proliferation than higher ones. 6-Benzylaminopurine (BAP) was superior to kinetin (KN) in shoot-tip proliferation. The optimum culture conditions for shoot-tip proliferation were achieved on MS medium supplemented with 0.25 mg/l 6-Benzylaminopurine (BAP) and 25 mg/l Salicylic acid (SA). The optimum culture conditions for callus formation were obtained on MS medium supplemented with 3 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 25 mg/l Salicylic acid (SA), while the optimum culture conditions for somatic callus formation were obtained on MS medium supplemented with 2.0 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 10 mg/l Salicylic acid (SA). The optimum culture conditions for rooting of shoots were obtained on MS medium supplemented with 0.25 mg/l of Indole-3-butyric acid (IBA) and 10 mg/l Salicylic acid (SA). The highest percentage of survival plants was obtained in the soil mixture supplied with 10 mg/l Salicylic acid (SA). Salicylic acid (SA) showed positive effect and good response on callusing, shooting and rooting of Z. spina-christi. Cultures received small amounts of salicylic acid were better than those lack it.

Manipulation of hemoglobin expression affects Arabidopsis shoot organogenesis

Over the past few years non-symbiotic plant hemoglobins have been described in a variety of plant species where they fulfill several functions ranging from detoxification processes to basic aspects of plant growth and post-embryonic development. To date no information is available on the role of hemoglobins during invitro morphogenesis. Shoot organogenesis was induced in Arabidopsis lines constitutively expressing class 1, 2 and 3 hemoglobins (GLB1, 2 and 3) and lines in which the respective genes were either downregulated by RNAi (GLB1) or knocked out (GLB2 and GLB3). The process was executed by culturing root explants on an initial auxin-rich callus induction medium (CIM) followed by a transfer onto a cytokinin-containing shoot induction medium (SIM). While the repression of GLB2 inhibited organogenesis the over-expression of GLB1 or GLB2 enhanced the number of shoots produced in culture, and altered the transcript levels of genes participating in cytokinin perception and signalling. The up-regulation of GLB1 or GLB2 activated CKI1 and AHK3, genes encoding cytokinin receptors and affected the transcript levels of cytokinin responsive regulators (ARRs). The expression of Type-A ARRs (ARR4, 5, 7, 15, and 16), feed-back repressors of the cytokinin pathway, was repressed in both hemoglobin over-expressors whereas that of several Type-B ARRs (ARR2, 12, and 13), transcription activators of cytokinin-responsive genes, was induced. Such changes enhanced the sensitivity of the root explants to cytokinin allowing the 35S::GLB1 and 35S::GLB2 lines to produce shoots at low cytokinin concentrations which did not promote organogenesis in the WT line. These results show that manipulation of hemoglobin can modify shoot organogenesis in Arabidopsis and possibly in those systems partially or completely unresponsive to applications of exogenous cytokinins.

Corolla of the Large-flowered Petunia (Petunia hybrida Vilm.) Cultivars Exhibit Low Endogenous Cytokinin Concentration through Enhanced Expression of the Genes Encoding Cytokinin Oxidases

We previously reported that cytokinin (CK) application enlarged the corolla of petunia (Petunia hybrida Vilm.). In this paper, the effect of the large-flowered trait of petunia caused by the Grandiflora (G) allele to CK biosynthesis was investigated. Concentrations of endogenous CK nucleobases (i.e., N6-(Δ2-isopentenyl)adenine (iP) and trans-Zeatin (tZ)), CK nucleosides (iP riboside and tZ riboside), and a CK glucoside (iP-7-glucoside) in the developing corolla were markedly lower in large-flowered cultivars with a Gg genotype than small- and medium-flowered cultivars with a gg genotype. We isolated the cDNAs encoding CK biosynthesis enzymes expressed in the young corolla, i.e., cDNAs of an adenosine phosphate-isopentenyltransferase gene (SHO), a CK riboside 5'-monophosphate phosphoribohydrolase gene (PhLOG), and two CK oxidase/dehydrogenase genes (PhCKX1 and PhCKX2). Expression levels of SHO and PhLOG in the developing corolla were not markedly different among small-, medium-, and large-flowered cultivars. However, expression levels of PhCKX1 and PhCKX2, which catalyze CK degradation through removal of the isoprenoid side chain, were much higher in large-flowered cultivars than in small- and medium-flowered cultivars. Therefore, low CK concentrations in large-flowered cultivars are very probably induced by the promoted expressions of PhCKX1 and PhCKX2, which may partially repress corolla enlargement.

Endogenous cytokinins, auxins and abscisic acid in Ulva fasciata (Chlorophyta) and Dictyota humifusa (Phaeophyta): towards understanding their biosynthesis and homoeostasis

Hormones are present in seaweeds, but little is known about their biosynthesis and regulation. The aim of this study was to investigate variation in concentration and composition of endogenous cytokinins, auxins and abscisic acid in two seaweeds collected over a 1 year period to gain a more complete picture of the types of hormones present during a range of environmental and developmental conditions. Ulva fasciata (Ulvales, Chlorophyta) and Dictyota humifusa (Dictyotales, Phaeophyta) were collected bimonthly from the inter-tidal zone at Rocky Bay, South Africa. Ethanol extracts of the samples containing a mixture of internal standards were purified using a combined DEAE-Sephadex-octadecylsilica column, followed by immuno-affinity chromatography and analysed by high-performance liquid chromatography–mass spectrometry to quantify different cytokinins, auxins and abscisic acid. cis-Zeatin, isopentenyladenine and their ribotide and riboside conjugates were the main cytokinins present in both U. fasciata and D. humifusa with low concentrations of trans-zeatin, dihydrozeatin and aromatic cytokinins. Very low concentrations of O-glucosides and riboside-O-glucosides and no N-glucosides were detected in any of the samples. Based on the cytokinins detected, we propose that as in higher plants, the ribotides are also the first intermediates formed during de novo biosynthesis in seaweeds and are subsequently converted to free-bases and ribosides. The only indole compounds detected in both species were free indole-3-acetic acid and indole-3-acetamide (IAM), suggesting that IAM is the intermediate in auxin biosynthesis. Endogenous abscisic acid was detected in most samples with levels in U. fasciata generally being higher than those measured in D. humifusa.

Cytokinin Import Rate as a Signal for Photosynthetic Acclimation to Canopy Light Gradients

Plants growing in dense canopies are exposed to vertical light gradients and show photosynthetic acclimation at the whole-plant level, resulting in efficient photosynthetic carbon gain. We studied the role of cytokinins transported through the transpiration stream as one of probably multiple signals for photosynthetic acclimation to light gradients using both tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We show that substantial variation in leaf transpiration parallels the light gradient in tobacco canopies and experimental reduction of the transpiration rate of a leaf, independent of light, is sufficient to reduce photosynthetic capacity in both species, as well as transcript levels of the small subunit of Rubisco (rbcS) gene in Arabidopsis. Mass spectrometric analysis of xylem sap collected from intact, transpiring tobacco plants revealed that shaded leaves import less cytokinin than leaves exposed to high light. In Arabidopsis, reduced transpiration rate of a leaf in the light is associated with lower cytokinin concentrations, including the bioactive trans-zeatin and trans-zeatin riboside, as well as reduced expression of the cytokinin-responsive genes ARR7 and ARR16. External application of cytokinin to shaded leaves rescued multiple shade effects, including rbcS transcript levels in both species, as did locally induced cytokinin overproduction in transgenic tobacco plants. From these data, we conclude that light gradients over the foliage of a plant result in reduced cytokinin activity in shaded leaves as a consequence of reduced import through the xylem and that cytokinin is involved in the regulation of whole-plant photosynthetic acclimation to light gradients in canopies.