Jiri Malbeck

O-Glucosylation of cis-Zeatin in Maize. Characterization of Genes, Enzymes, and Endogenous Cytokinins

trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize (Zea mays) gene (cisZOG1) encoding an O-glucosyltransferase specific to cis-zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922–5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression ofcisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K m values of 46 and 96 μm, respectively, for cis-zeatin and a pH optimum of 7.5. Other cytokinins, including N6-(Δ2-isopentenyl)adenine, trans-zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and theirO-glucosides were detected in all maize tissues, with immature kernels containing very high levels of theO-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.

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

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

Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation

To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.

Evidence for Importance of tRNA-Dependent Cytokinin Biosynthetic Pathway in the Moss Physcomitrella patens

To study cytokinin biosynthesis, we characterized a temperature-sensitive cytokinin-overproducing mutant, oveST25, of the moss Physcomitrella patens with respect to changes in cytokinin content during thermal induction in comparison to wild type. Our findings, based on combined liquid chromatography-mass spectrometry (LC-MS) analyses, show that thermoinduction caused a strong increase of extracellular N6-(Δ2-isopentenyl)adenine (iP), N6-(Δ2-isopentenyl)adenosine (iPR), cis-zeatin (cZ), cis-zeatin riboside (cZR) and its O-glucoside cZROG in oveST25. In contrast, no significant changes were measured in the wild type. To investigate the relevance of tRNA for cytokinin production in Physcomitrella, we determined cytokinins in tissue and culture medium as well as in tRNA hydrolysates. The analysis of cytokinins from whole-culture extracts of wild type revealed 56% of iP-type, 32% of cZ-type, and 11% of trans-zeatin (tZ)-type forms. In tRNA, 90% of cytokinins were represented by cZ-type and 8% by iP-type forms; tZ-type cytokinins were found only in trace amounts. The finding that the major free cytokinins are, albeit with altered proportions, also major forms in tRNA is compatible with the hypothesis of a strong tRNA-mediated biogenesis of cytokinins in this plant. Our RT-PCR-based studies on the expression of the tRNA-IPT gene, PpIPT1, revealed enhanced transcription levels in the cytokinin-overproducing oveST25 mutant at the inducing temperature of 25°C, but not at noninducing conditions (15°C). A wild-type transgenic line with cytokinin deficiency due to heterologous cytokinin oxidase/dehydrogenase overexpression (AtCKX2) also exhibited enhanced PpIPT1 expression levels, indicating that cytokinin deficiency might upregulate tRNA-mediated cytokinin biosynthesis. The evidence that the tRNA-mediated pathway might be mainly responsible for biosynthesis of isoprenoid cytokinins in Physcomitrella is strongly supported by the recent release of the Physcomitrella genomic sequence where only tRNA-IPTs but no adenylate-IPTs are present.

Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity.

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.

Modification of cytokinin levels in potato via expression of the Petunia hybrida Sho gene.

The Sho gene from Petunia hybrida encodes an enzyme for cytokinin synthesis. Here we report on the effects of Shogene expression on potato development. In contrast to transgenic potato expressing the Agrobacterium ipt gene, moderate Sho expression resulted in sufficient root development that allowed the cultivation of the Sho transformants in soil. The most pronounced effects detectable in these lines were an enhanced shoot production, delayed tuber formation, significant reduction in tuber size, and inhibition of tuber dormancy. Sho expression predominantly associated with a strong increase in 2iP glucosides, accompanied by an increase in zeatin glucosides in lines with very high Sho expression levels. The data demonstrate that it is possible to produce viable plants with enhanced cytokinin levels via constitutive Sho expression, which allows an assessment of cytokinin effects in all organs.

AtSOFL1 and AtSOFL2 Act Redundantly as Positive Modulators of the Endogenous Content of Specific Cytokinins in Arabidopsis

Background Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited. Methodology/Principal Findings Here, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2s overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N6-(Δ2-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays. Conclusions/Significance Taken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.