Presence of 2-methylthioribosyl-trans-zeatin in Azotobacter vinelandii tRNA.

Hydroxylated cytokinin, 2-methylthio-N6-(4-hydroxy-3-methylbut-2-enyl) adenosine, was found in the tRNA of Azotobacter vinelandii. This cytokinin had the trans configuration, unlike the cis configuration reported for that from other bacteria. Culture-condition-dependent changes in the content of this thiocytokinin and a few other thionucleosides in the tRNA of this bacterium have been observed.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin

Zeatin is a naturally occurring cytokinin. Biosynthesis and metabolism studies of zeatin have been directed mostly at the trans isomer, although cis-zeatin and its riboside occur as major components in some plant species. It is not known whether parallel regulatory pathways exist for the two isomers. Based on the sequence of the gene ZOG1 encoding a trans-zeatin O-glucosyltransferase from Phaseolus (EC 2.4.1.203), a cis-zeatin-specific O-glucosyltransferase was isolated from maize. This gene, cisZOG1, contains an ORF of 1,401 nucleotides encoding a protein of 51.1 kDa with 41% identity to the Phaseolus ZOG1 protein. Unexpectedly, the maize enzyme recognizes as substrates cis-zeatin and UDP-glucose but not cis-ribosylzeatin, trans-zeatin, or trans-ribosylzeatin. This finding indicates the existence of cis-specific regulatory elements in plants and suggests that cis-zeatin and derivatives may be more important in cytokinin homeostasis than currently recognized.

Unique presence of 2-methylthio-ribosylzeatin in the transfer ribonucleic acid of the bacterium Pseudomonas-Aeruginosa

Analysis of 35S labled nucleosides prepared from tRNA of Pseudomonas aeruginosa by phosphocellulose column chromatography, thin layer chromatography and Sephadex LH-20 column chromatography revealed the presence of 2-methylthioribosylzeatin in it. 2iPA, 6-(3-methyl-2-butenylamino)-9-β-D-ribofuranosyl purine; ms-2iPA, 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine; ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-β-D-ribofuranosylpurine; ribosyl-trans-zeatin, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-β-D-ribofuranosylpurine; ms-ribosylzeatin, 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine; s4U2, 4-thiouridine; s2U*, 5-methylaminomethyl-2-thiouridine; s2C, 2-thiocytidine; TLC — thin layer chromatography.

Unique presence of 2-methylthio-ribosylzeatin in the transfer ribonucleic acid of the bacterium Pseudomonas-aeruginosa

Analysis of 35S labled nucleosides prepared from tRNA of Pseudomonas aeruginosa by phosphocellulose column chromatography, thin layer chromatography and Sephadex LH-20 column chromatography revealed the presence of 2-methylthioribosylzeatin in it. 2iPA, 6-(3-methyl-2-butenylamino)-9-β-D-ribofuranosyl purine; ms-2iPA, 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine; ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-β-D-ribofuranosylpurine; ribosyl-trans-zeatin, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-β-D-ribofuranosylpurine; ms-ribosylzeatin, 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine; s4U2, 4-thiouridine; s2U*, 5-methylaminomethyl-2-thiouridine; s2C, 2-thiocytidine; TLC — thin layer chromatography.

Hormones andCuscuta development: In vitro induction of haustoria by cytokinin and its inhibition by other hormones

Cytokinins induced haustoria formation in excised 10-mm segments ofCuscuta vine, the subapical 25-to-50-mm region being most responsive, producing a mean of 4–6 haustoria per segment. The order of effectiveness of cytokinins continuously applied (72 h) was 6-benzylaminopurine (BA) ges isopentenyladenine (iP) Gt zeatin (Z). Ribosides of BA and Z were as effective as the bases, whereas riboside of iP ([9R]iP) was half as effective as iP. Haustoria induction was influenced by weather and seasonal conditions at the time of vine collection; materials obtained on warm, sunny days responded better than those obtained on rainy, cloudy, or cool days. Haustoria were induced equally well all around the segment, and no thigmostimulus was needed for induction. p ]A 10-min pulse of 100 mgrM BA induced half as many haustoria as a 60-min pulse or continuous application of BA. White light inhibited haustoria induction elicited by a short (30-min) pulse of BA, whereas a longer (120-min) BA application overcame this light inhibition. Auxins (IAA or NAA, 1–10 mgrM), gibberellin (GA3, 1–10 mgrM), ethylene (as ethrel, 10–100 mgrM), and abscisic acid (ABA, 100 mgrM) were individually inhibitory (60–80%) with respect to haustoria induction when given continuously with 50 mgrM BA. A 60-min pulse of auxins (10 mgrM), GA3 (100 mgrM), or ethrel (10 mgrM), given at various time intervals during or after a 60-min pulse of 100 mgrM BA, showed that inhibition was maximal (70–95%) between 4 and 16 h of BA application and negligible (GA3) or much reduced (auxin, ethrel) at 20 h, indicating a ldquocommitmentrdquo to haustoria formation by this time.

Morphogenesis and plant regeneration from cotyledonary cultures of Eucalyptus

Callus cultures were established from hypocotyls and cotyledons derived from young seedlings of Eucalyptus citriodora. Successful plantlet production from cotyledonary callus was achieved within 6 weeks on Murashige and Skoog's basal medium supplemented with zeatin (1 mg/l) and indoleacetic acid (0.2 mg/l). Leaf and shoot callus obtained from one-year-old plants did not differentiate. Results reported contribute to defining optimal conditions for callus growth and plantlet formation

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Isolation and characterization of a cytokinin-binding protein from cucumber cotyledons

A protein which binds specifically to [3H]-zeatin has been isolated from cucumber cotyledons by chromatographic techniques. Its binding to [3H]-zeatin was inhibited remarkably by the addition of non-radioactive cytokinins and the order of inhibition was zeatin > -zeatin riboside > N6-(Delta2-isopentenyl)adenine > N6-(Delta2-isopentenyl)adenosine > N6-benzyl-adenosine > kinetin riboside. This protein behaved as a soluble protein with an apparent molecular size of 43,000 daltons on gel filtration through calibrated Sephadex G-100 column. The dissociation constant, Kd, of the protein-zeatin complex was about 4 × 10–7 M.

Respuesta a la sequía de Pinus radiata D. Don y su implicación en los procesos de tolerancia

160 p. (Bibliogr. 141-160)

诱导风信子（Hyacinthus orientalis L.）同一花被外植体不同部位细胞分化花芽的研究--外源激素的作用及内源激素的变化

诱导风信子（Hyacnthus orientalis L．）同一花被外植体上不同部位细胞分化花芽，从外源激素的作用和内探激素的变化探讨细胞脱分化启动的原因，研究了不同外源激素的组合下同一花被不同部位花芽分化的诱导频率：测定了花被上、中，下三部位切块离体培养前后的内源IAA和Z+ZR的含量;在此之前研究了GC-MS．MIM内标法测定微量植物材料内源IAA含量的可行性． GC-MS．MIM内标法是测定微量（0.5-1g）植物材料内源IAA含量的一种比较理想的方法，所需材料量一般为0.58．这一方法的材料前处理采用粗提液用C18Sep-pak柱初步分离纯化．HPLC进一步纯化，能获得纯度高的样品，且操作简便，省时省力． 风信子同一花被不同部位细胞均能分化花芽．当培养基中附加2.0mg/l Zeatin或2.0mg/16-BAP时，随着外探IAA浓度从0升高到10.0mg/l．捆胞分化花芽的部位从花被下部向上部移动。 离体培养前后同一花被上、中、下三部位内源IAA和Z+ZR含量测定结果表明．风信子同一花被内源IAA含量是上部最高，下部次之，中部最低，而内源Z+ZR含量从上向下依次增加;在附加不同外源激素的MS培养基上培养3天后，同一花被上，中、下三部位内源IAA和Z+ZR含量部有一定的变化。

拟南芥有性生殖模式和叶片特性对大气CO2浓度变化的响应

预计到本世纪末，大气CO2浓度将会增加到540～970ppm，大气CO2浓度升高所引起的全球气候变化已经受到广泛的关注。植物生长依赖CO2，并且对大气CO2浓度升高在结构和生理上产生响应。目前已有大量报道，从生态系统、群落、种群、个体、器官、组织、生理以及生化等水平上研究高浓度CO2所对植物产生的影响。但是有关高浓度CO2对植物有性生殖影响的报道却很少，同时多数实验均建立在短期的生殖响应，忽视了植物在长期高CO2浓度下具有的反馈作用和CO2浓度变化对植物的驯化作用。植物有性生殖与其生态适应性和农作物籽粒产量的关系极为密切;同时，植物有性生殖特性的变化，也可作为预测植物对全球气候变化响应的重要指标之一。为此，利用高浓度CO2对植物进行长期选择实验将很有必要。研究结果将为预测未来大气CO2浓度增加的条件下陆地生态系统的演变趋势、全球变化对植物有性生殖响应的方式和机制提供新的思路和有效方法。 　　在本研究中，我们以模式植物拟南芥(Arabidopsis thaliana)作为实验材料，利用370和700ppm CO2对其进行连续8个世代处理，首先研究高浓度CO2对每一个世代的拟南芥有性生殖特性的影响，然后比较各个世代中各种生殖特性指标变化的规律，从细胞、组织和个体尺度上揭示拟南芥有性生殖对全球变化的响应模式。此外，在700ppm CO2处理下，我们对拟南芥叶片生理、生化以及结构的变化进行了相关研究。两部分研究结果及主要结论如下： 　　首先，在每一个世代中，与370ppm CO2相比较，700ppm CO2处理显著促进了拟南芥开花，缩短生长周期，增加花、角果及种子等生殖的产量，降低种子N含量，提高种子C/N比、种子千粒重以及生殖生物量所占总生物量的比例等，而对种子萌发率、角果所含种子数目以及角果长度则无显著影响。但是， 通过对相同CO2浓度处理条件下，不同世代之间的研究结果比较发现，不同世代之间相关的生殖生物学指标并无显著差异。 　　其次，高浓度CO2显著降低叶片气孔密度、气孔指数、气孔导度以及蒸腾速率。在高浓度CO2处理下，叶肉细胞中叶绿体数目、叶绿体宽度和表观面积、淀粉粒大小和数量、叶片和细胞壁厚度等都显著增加，但是基粒内囊体膜的数量却显著下降。叶片中碳水化合物如可溶性总糖、淀粉以及纤维素含量在高浓度CO2下分别显著增加71.9%、78.7% 和 22.3%。此外，在高浓度CO2处理下，叶片中多数激素如如吲哚乙酸(indole-3-acetic acid, IAA)、赤霉素(gibberellin, GA)、玉米素核苷(zeatin riboside, ZR)、二氢玉米素核苷(dihydrozeatin riboside, DHZR)和异戊烯基腺苷(isopentenyl adenosine, iPA)均都显著地增加，而脱落酸(abscisic acid, ABA)含量却有所下降。最后，叶片中各种矿物质元素含量如N、P、K、Ca和Mg等含量在高浓度CO2处理下也都显著下降，而C/N比增加24.8％。 　　以上结果表明： 　　(1) 在每一个世代中，700ppm CO2处理对拟南芥各种有性生殖特性具有显著的影响，但是高浓度CO2处理对植物所引起的效应在多个世代以内并不能够传递给后代，所以在多个有性生殖世代内，高浓度CO2处理对植物生长、生殖没有驯化作用。 　　(2) 在高浓度CO2处理下，拟南芥叶片中叶绿体超微结构的变化，可能主要是由于叶绿体中淀粉粒数量和体积大小显著增加而引起。 　　(3) 在高浓度CO2处理下，由于拟南芥叶片内与促进细胞分裂与伸长的激素含量显著增加，从而对拟南芥植株生长发育速率的提高起了重要的作用。 　　(4) 拟南芥生长在高浓度CO2条件下，其叶片中各种矿质元素含量（如N、P、K、Ca和Mg）均显著降低，究其原因可能是，第一由于叶片中碳水化合物含量的显著增加而对矿物质元素具有稀释作用;第二由于蒸腾速率下降，引起矿质元素从根部随着蒸腾流运输到地上部分的含量相应减少。 　　

Characterization and analysis of the exogenous application of selected phytohormones on C. reinhardtii metabolism

Gemstone Team Genes to Fuels

A breakthrough in lupin biotechnology: prolific protocolonisation in recalcitrant white lupin (Lupinus albus) triggered by bovine serum albumin

Six nutrient formulations were studied for their efficacy in inducing mitosis in white lupin seedling cotyledon protoplasts of which the formulations of Schafer-Menuhr & Sturmer (AS) and Kao (K8p) were found to be superior over the other four when supplemented with 6-benzylaminopurine and alpha-naphthaleneacetic acid (alpha-NAA). An unltrafiltration treatment of K8p increased mitotic frequency by 130% when compared with the untreated control. Medium enrichment with 0.2% bovine serum albumin (BSA) brought about a dramatic 1341% rise in protoplast division in comparison with BSA-free medium but only when the enrichment was carried out in Kao and Michayluk (KM8p) background containing 2, 4-dichlorophenoxyacetic acid, alpha-NAA and zeatin. A higher number of protocolonies (each proliferating from single protoplast following multiple divisions) were seen in 0.4% BSA. With this breakthrough in white lupin protoplast research, it is now possible to reproducibly obtain protocolonies that was hitherto not possible.

Adventitious root formation in anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as 'difficult'- or 'easy-to-root' when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be 'difficult' precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting. In this study we used softwood cuttings of cashew (Anacardium occidentale), a species considered as 'difficult-to-root'. We aimed to test the hypothesis that speed, and extent of early rooting, is critical in determining success with this species; and that the potential to form adventitious roots will decrease with time in the propagation environment. Using two genotypes, initial rooting rates were examined in the presence or absence of exogenous auxin. In cuttings that formed adventitious roots, either entire roots or root tips were removed, to determine if further root formation/development was feasible. To investigate if subsequent root responses were linked to phytohormone action, a number of cuttings were also treated with either exogenous auxin (indole-3-butyric acid-IBA) or cytokinin (zeatin). Despite the reputation of Anacardium as being 'difficult-to-root', we found high rooting rates in two genotypes (AC 10 and CCP 1001). Removing adventitious roots from cuttings and returning them to the propagation environment, resulted in subsequent re-rooting. Indeed, individual cuttings could develop new adventitious roots on four to five separate occasions over a 9 week period. Data showed that rooting potential increased, not decreased with time in the propagation environment and that cutting viability was unaffected. Root expression was faster (8-15 days) after the removal of previous roots compared to when the cuttings were first stuck (21 days). Exposing cuttings to IBA at the time of preparation, improved initial rooting in AC 10, but not in CCP 1001. Application of IBA once roots had formed had little effect on subsequent development, but zeatin reduced root length and promoted root number and dry matter accumulation. These results challenge our hypothesis, and indicate that rooting potential remains high in Anacardium. The precise mechanisms that regulate the number of adventitious roots expressed, remain to be determined. Nevertheless, results indicate that rooting potential can be high in 'difficult-to-root' species, and suggest that providing supportive environments is the key to expressing this potential. (c) 2006 Elsevier B.V. All rights reserved.