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  1. 1. ADVANCE IN ENVIRONMENTAL STRESSES: Term paper Topic: Cytokinins’ active form and their roles in plant morphology and develop- ment 972004206 2010/01/21 Selected papers: T. Werner, V. Motyka, M. Strnad, and T. Schmülling (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98: 10487-92 T. Kuroha, H. Kato, T. Asami, S. Yoshida, H. Kamada and S. Satoh (2002) Zeatin, discovered by Letham, 1973 A trans-zeatin riboside in root xylem sap negatively regulates adventitious root formation on cucumber hypocotyls. J. Exp. Bot. 53: 2193-200 Abstract: During the presentation of the previous research paper (Š. Vyroubalová et al., 2009) A question was given: Whether cy- tokinin promotes plant growth or suppress it, and what forms are cytokinins' active forms? In a search for a better under- standing of this plant hormone's physiological role in plant de- velopment, literatures published after 2000 have been surveyed with various keywords (cytokinin, regulation, active forms and growth), and two research papers have been selected for the term paper to address these two questions. A brief introduction to the two research papers will first be given, then the correla- tion of their results between the topic. (Contents that is adapted (i.e., copied and pasted) will be in italic fonts.) Abbreviations CK cytokinin CK cytokinin oxidase I. Introduction X iP N6-(∆2- isopentenyl)adenine T. Werner et al., 2001 BA benzyladenine tZ trans-zeatin There are two papers selected for the term paper, a brief introduction tZR trans-zeatin riboside to the experiments conducted in (T. Werner et al., 2001) will first be cZ cis-zeatin given. cZR cis-zeatin riboside SA shoot apical meristem M
  2. 2. CK's regulation effect on plant growth is ana- and roots are compared with wildtypes’, i.e., plants lyzed by an indirect novel approach using AtCKX with higher CK content (Fig. 4). transgenics which down-regulates CK levels. That is, the AtCKX genes are first identified by a AtCKX transgenics BLAST search, cloned, positioned under the con- Shoot (Leaves) (Fig. 4 A-D) trol of a constitutively expressed 35S promoter, SAM, size of SAM (-)smaller which constantly express CKX and down-regulates SAM, size of cells (X)unchanged CK level in vivo. Through this approach, CK's ef- xylem, phloem (-)smaller fect can be seen with an endogenous control of CK leaf epidermis cells (+)bigger level, rather than exogenous CK addition, e.g. BA incubation. leaf parenchyma cells (+)bigger Root tips (Fig.4 E-G) The followings are conducted in this research: cell numbers of root tips (+)greater (Materials and Methods) cell size of root tips (+)bigger radial root pattern (X)unchanged 1. Gene Cloning Source: Original data from Fig. 4 (T. Werner et al., 2001) 2. Plant Transformation and Culture 3. RNA Preparation and Blot Analysis 4. Histological Analysis As the result of down-regulated cytokinins, a 5. Morphology and Phenotype Analysis tendency is clearly seen that without CK, stem 6. Quantitative Analysis of Cytokinin Oxidase growth seems to be repressed, and root growth pro- 7. Quantitative Analysis of Cytokinin Content moted. Yet it can be seen that the regulation on growth is not so straightforward as simply being The first two are the preparation of AtCKX positive or negative/ up or down, e.g., cell size and transgenics and the following RNA preparation and cell cell numbers have to be taken into account si- Blot Analysis is to select the positive clones, in this multaneously and separately. As an interesting ex- case, AtCKX1-28, 8, and 15 (fig. 1A). The essen- ample, compare the macroscopic (Fig. 3) and micro- tial results of this paper lies in the latter four, histo- scopic observations: analysis, morphology and Phenotype Analysis and the quantitative analysis of CKX and CK. In Fig. 3 (macroscopic), root is longer while stem is shorter; however, in p.10490 (T. Werner et (Note: There are four AtCKX genes, AtCKX1-4. al., 2001) it has been written: 28, 8, and 15 denotes different clones.) In the AtCKX transgenics, the final length of cells In the macroscopic analysis (Morphology and in the stem is not reduced, and the final length of root cells was slightly decreased (149.7 ± 31.7 µM phenotype analysis), a clear trend can be seen: in clone AtCKX1-50 versus 167.0 ± 32.0 µM in With the constitutively expressing CKX enzymes wildtype; n = 100), indicating that differences in and the subsequent low CK level, shoot growth are cell growth did not contribute to, or even par- clearly suppressed while root shows an completely tially compensate for, altered growth of stem or opposite effect 17 days after germination (Fig. 3) root. which is a noteworthy phenomenon. There are few AtCKX transgenics points that could be deduced from this paper: Seedling, 17 days after germination Root length (primary root) longer 1. Macroscopically, CK promotes stem growth, and At flowering stage suppress root (including adventitious root) Root length longer growth. Source: Original data from Fig. 3 (T. Werner et al., 2001) 2. Cell size, cell number, and organ growth should be considered separately, as the example just In the histological-analysis, AtCKX transgen- given in the last paragraph. Even cell length and ics’ transverse and longitudinal sections of leaves cell width should be separately considered. 2
  3. 3. These two results, taken together, indicates T. Kuroha et al., 2002 that ZR might be the sole inhibitory factor in the sap as Z is present in the sap with an inadequate (Materials and Methods) concentration to cause the inhibitory effect ob- served. 1. Plant materials and chemicals 2. Collection of root xylem sap 3. Fractionation of the inhibitory factor in root II. Comparison of the selected xylem sap papers 4. Identification and quantification of the inhibi- tory factor Research T. Werner et al., T. Kuroha et al., Š. Vyroubalová 5. Induction of the formation of adventitious roots papers 2001 2002 et al., 2009 Cumcumber Tabacco (Solana- Subjects and squash Maize (Poaceae) In this paper, xylem sap was utilized to exam- ceae) (Cucurbitaceae) ine cytokinins' effect on adventitious root forma- Evaluate CK Evaluate CK Complete char- tion. For a source of xylem sap, squash plants are effects by manipu- effects by add- acterization of lating endoge- ing CK exoge- the whole CK- used as they produce abundant xylem sap (which nous CK level nously (CK related genes was later found to contain tZR at the concentration (CK level ↓) level ↑) of 2×10 -8M). The cucumbers are then incubated in the purified xylem sap exudate collected from squash and are followed by the induction of the formation of adventitious roots. A simple comparison of the two selected papers, note that one paper up-regulates CK level exoge- As is implied, this paper is published in the nously while one, oppositely and coincidentally, Journal of Experimental Botany, most of its con- down-regulates CK level endogenously. tent focuses upon the experiments of purification and identification processes using HPLC system- III. Conclusions based reverse/normal phase chromatography and LC/MS/MS (liquid chromatography/ mass spec- In this section, some important papers on the troscopy/ mass spectroscopy)to purify and identify research of CK regarding this topic (CK’s roles in the inhibitory factor (tZR). However, in spite of the plant morphology and development) will be in- experimental processes, I will be focusing on its cluded and discussed with the two selected papers. final results which are relevant to my subject Returning first to my subject: (Whether cytokinin promotes plant growth or sup- press it, and in what forms will cytokinins be bio- 1. CK’s role in plant morphology and development logically active? ). In Table. 1 (Cytokinin levels in 2. CK’s active forms the xylem sap collected from squash roots) and Fig. 6 (The inhibitory effect of cytokinins, ZR, Z CK’s role in plant morphology and development and BA), there are few points that could be de- duced from this paper: From (T. Werner et al., 2001), we know that 1. Both tZ and cZ was found in the xylem sap macroscopically, CK promotes stem growth, and with concentration at the order of 10-10 (Table. suppress root (including adventitious root) growth. 1). This gives only a very small part of the answer, in 2. However, as compared with Fig. 6, the inhibi- that trying to give a complete picture for this “puz- tory effect on adventitious root formation of zle” might actually be far beyond the scope of the zeatin plummeted at the concentrations below term paper and time may not permit me to do so. 10-8 (ZR, Z and BA converged at promotion However, I will include some other informations rate=0, at the concentration of 10-10 ). from other papers which will shed light on the com- plex questions. 3
  4. 4. Active forms of CK 4. H. Yamada, T. Suzuki, K. Terada, K. Takei, K. Ishikawa, K. Miwa, T. Yamashino and T. Mizuno (2001) The Arabidopsis AHK4 Histidine Kinase is a Cytokinin-Binding Receptor that In the “quest” to determine the active forms of Transduces Cytokinin Signals Across the Membrane. Plant Cell CK, it has been quite difficult because various CK Physiol. 42: 1017-23 forms do interconvert in vivo with the help of en- 5. T. Kamada-Nobusada, H. Sakakibara (2009) Molecular basis for zymes, e.g. trans-cis zeatin isomerase. A paper has cytokinin biosynthesis. Phytochemistry 70: 444-9 been published titled “The Arabidopsis AHK4 His- 6. F. Skoog and C. O. Miller (1957) Chemical regulation of growth tidine Kinase is a Cytokinin-Binding Receptor that and organ formation in plant tissue cultured. In vitro. Symp. Soc. Transduces Cytokinin Signals Across the Mem- Exp. Biol. 11: 118-31 brane.” (Yamada et al., 2001) 7. K. Wilson, J. Walker (2005) Principles and Techniques of Bio- chemistry and Molecular Biology, 6th ed., Cambridge University What it means to discover a CK receptor is, with Press: New York. pp. 430-34, 500-14 (Chapters on chromatogra- the aid of the receptor, we shall be able to find the phy and Temden Mass Spectroscopy) active forms of CK by designing an experiments that 8. L. Taiz, and E. Zeiger (2006) Plant Physiology, 4th ed., Sinauer determine which forms of CK are actually binding to Associates, Inc.: Massachusetts pp. 543-67 (Chapter 21: Cytoki- nin: Regulators of cell division) the receptor. Therefore, this question can then be ad- dressed directly. Besides, in various review articles on CK biosyn- thesis, it had been mentioned many times that the active forms are the free bases (Š. Vyroubalová et al., 2009; T. Kamada-Nobusada and H. Sakakibara, 2009). Lastly, in regard to what the active forms of CKs are, one should consider: 1. Organism species (different species, different receptors) 2. The co-regulation of CKs with other plant hor- mones such as the auxin-cytokinin hypothesis of plant morphogenesis (F. Skoog and C.O. Miller, 1957) and ABA (Š. Vyroubalová et al., 2009)(p. 434, line 59). 3. Dosage-dependent CK signaling (F. J. Ferreira and J. J. Kieber, unpublished) IV. References 1. T. Werner, V. Motyka, M. Strnad and T. Schmülling (2001) Regulation of Plant Growth by Cytokinin. Proc. Natl. Acad. Sci. USA 98: 10487-92 2. T. Kuroha, H. Kato, T. Asami, S. Yoshida, H. Kamada and S. Satoh (2002) A trans-Zeatin Riboside in Root Xylem Sap Nega- tively Regulates Adventitious Root Formation on Cucumber Hypocotyls. J. Exp. Bot. 53: 2193-200 3. Š. Vyroubalová, K. Václavína, V. Turečkova, O. Novák, M. Šmehilová, T. Hluska, L. Ohnoutková, I. Frébort and P. Gal- uszka (2009) Characterization of New Maize Genes Putatively Involved in Cytokinin Metabolism and their Expression during Osmotic Stress in Relation to Cytokinin Levels. Plant. Physiol. 151: 433-47 4
  5. 5. A gene encoding CK oxidase was first identi- fied in maize (Houba-Herin et al., 1999; Morris et al., 1999) 5