Title of Invention

A Process for Isolating Cytokinin Oxidase Nucleotide Sequence and uses thereof

Abstract The present invention relates to the emergence of the Cytokinin oxidase gene from the RASI variety of rice seedlings under salt stress. It also contemplates the role played by cytokinin oxidase in plant catabolism.
Full Text Form 3A
The Patents Act, 1970
Complete Specification (See Section 10)
1. Title of the Invention "Accumulation of Cytokinin Oxidase In Rice Under Salt Stress Protecting Plant Development".
2. Name of the Inventor/Applicant: Avestha Gengraine Technologies Pvt. Ltd., an Indian Company, registered under the provisions of The Companies Act, 1956, with its Office at "Discoverer", 9th Floor, Unit 3, International Tech Park, Whitefield Main Road, Bangalore - 560 066, Karnataka, India
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:
Field of Invention
The present invention relates to the emergence of the Cytokinin oxidase gene from the RASI variety of rice seedlings under salt stress. It also contemplates the role played by cytokinin oxidase in plant catabolism.
Prior Art
Regulation of active cytokinin levels in planta may occur through the control of synthesis through irreversible conjugation to inactive glucosides or through irreversible inactivation (Jameson, 1994). In many plant tissues oxidative cytokinin degradation appears to be the major pathway for cytokinin inactivation (Armstrong, 1994). Maize tissues have historically been shown to have potent cytokinin degrading activities and cytokinin oxidase activity have been partially purified from maize kernels and seedlings (Whitty & Hall, 1974; McGaw & Horgan, 1983; Burch & Horgan, 1989; Horgan et al., 1990).
US patent 6,229,066 describes an isolated protein which exhibits cytokinin oxidizing activity selected from the group consisting of SEQ. ID No. 1, a protein having an amino acid sequence which includes the amino acid sequence of SEQ. ID No. 1, a protein having an amino acid sequence which includes a portion of the amino acid sequence of SEQ. ID No. 1, the included portion being at least about 20 amino acid residues in length and conferring the cytokinin oxidizing activity on the protein, and a protein including an amino acid sequence with at least about 65% sequence identity to SEQ. ID No. 1, the remainder of amino acid residues being conservatively substituted. Nucleic acids encoding proteins which exhibit cytokinin oxidizing activity and related products and methods are also disclosed.
US patent application 20030074698 describes methods for stimulating root growth and/or enhancing the formation of lateral or adventitious roots and/or altering root geotropism comprising expression of a plant cytokinin oxidase or comprising expression of another protein that reduces the level of active cytokinins in plants or plant parts. Also provided by the present invention are methods for increasing seed size and/or weight, embryo size and/or weight, and cotyledon size and/or weight. The methods comprise expression of a plant cytokinin oxidase or expression of another protein that reduces the

level of active cytokinins in plants or plant parts. The invention also relates to novel plant cytokinin oxidase proteins, nucleic acid sequences encoding cytokinin oxidase proteins as well as to vectors, host cells, transgenic cells and plants comprising said sequences. The invention also relates to the use of said sequences for improving root-related characteristics including increasing yield and/or enhancing early vigor and/or modifying root/shoot ratio and/or improving resistance to lodging and/or increasing drought tolerance and/or promoting in vitro propagation of explants and/or modifying cell fate and/or plant development and/or plant morphology and/or plant biochemistry and/or plant physiology.
It was found that active cytokinin concentration decreases dramatically when Zea mays seedlings are exposed to low temperatures. Part of this decrease is the result of the activity of zeatin O-glycosyltransferase, which catalyze the formation of storage and stable form of cytokinin, zeatin O-glycosides. Cytokinin oxidase, which irreversibly deactivates zeatin, is also important in the regulation of the endogenous cytokinin levels in heat stressed plants and during normal development. Preliminary results showed that the cytokinin oxidase activity increased in root tips and shoot tips of Zea mays when seedlings (3-d-old, 25 OC) were cold stressed at 4 OC for 3 days. (Plant Biology 2000)
Plant cytokinins are a class of plant hormones which, when combined with auxin, control cell division, promote shoot development from callus, release lateral buds from dormancy, and regulate plant structure and growth in a variety of ways. The naturally occurring active cytokinins in most higher plants are free-base zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) (hereinafter Z), and its 9-riboside (hereinafter ZR). Plant tissues normally contain, therefore, Z, ZR, and smaller amounts of N.sup.6 -(.DELTA..sup.2 -isopentenyl)adenine (hereinafter, iP) derived from biosynthetic precursors. Elevated cytokinin levels are associated with the development of seeds in higher plants, and have been demonstrated to coincide with maximal mitotic activity in the endosperm of developing maize kernels and other cereal grains. Exogenous cytokinin application (via stem injection) has been shown to directly correlate with increased kernel yield in maize. In addition, plant cells transformed with the ipt gene from Agrobacterium tumefaciens (encoding a dimethylallylpyrophosphate:5'-AMP transferase capable of increasing cellular production of Z and ZR) showed increased growth corresponding to an increase in endogenous cytokinin levels upon induction of the enzyme. Thus, given the biosignificance of cytokinins to the growth of plants, the ability to manipulate cytokinin levels in higher plant cells is of great commercial and scientific interest.
The action of cytokinin oxidase is a major method of effective cytokinin catabolism in plant cells. This inactivation of cytokinin is accomplished by the oxidative removal of the side chain from cytokinin free bases (or their ribosides) in the presence of molecular oxygen. Although the exact chemical mechanism for this reaction is unknown, it is suspected that the enzyme is reduced during the deprotonation of iP to N.sup.6 -(.DELTA..sup.2 -isopentenylimino)purine. The purine is then hydrolysed into adenine and intermediate 3-methyl-2-butenal. While the electron acceptor responsible for reoxidizing the reduced enzyme in plant cells is not known, molecular oxygen can do so in vitro. Alternatively, the reduced enzyme may be reoxidized in vitro by intermediates such as Cu.sup.+2 /imidazole complexes or the artificial electron acceptor dichlorophenolindophenol (DCPIP).
Cytokinin oxidases are known to remove cytokinins from plant cells after cell division, and have also been postulated to be involved in the senescence process. Cytokinin oxidase activities have been shown to positively correlate to the mitosis of endosperm cells in maize kernels, along with the increase in natural cytokinin concentrations. Oxidase activity increases shortly after the increase in endogenous cytokinin levels. A similar correlation was demonstrated with artificially increased

cytokinin levels in transgenic tobacco. Thus, expression of cytokinin oxidases is thought to be involved in the maintenance of hormonal homeostasis in developing plant cells. Because cytokinin oxidases appear to be substrate-inducible, they act in a negative regulatory fashion to reduce elevated cytokinin levels back to basal values. This substrate induction of cytokinin oxidase activity is a significant barrier to potential commercial applications, which attempt to manipulate cytokinin levels in transgenic plants through increased cytokinin production.
Cytokinin oxidases have been discussed for a number of plant species, including Vinca rosea, beans (Phaseolus vulgaris and lunatus), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), Dianthus caryophyllus, soy (Glycine max), and maize (Zea mays). All of these plant cytokinin oxidases have a similar substrate preference for iP and Z, but show limited or no reactivity with bulky, reduced, or aromatic side chain cytokinins. These enzymes show substantial variation in both specific activity and molecular weight. This is thought to be linked to the occurrence of glycosylated and unglycosylated variants of the protein, both between and within species.
In the case of the glycosylated cytokinin oxidase, the heavily glycosylated protein may present a carbohydrate-rich surface, preventing antibody formation against peptide epitopes. The glyco-epitopes to which antibodies are raised under these conditions are non-specific, and may prevent isolation of the protein, or clones containing the gene which encodes it, via immuno-chromatography or other immunology-based means. An earlier reported attempt to isolate the gene for maize cytokinin oxidase (ckxl) by immunoscreening of maize cDNA library expression products (Burch, 1992) was unsuccessful. The majority of cytokinin oxidase activity was detected in maize reproductive tissue. A previous study established a temporal co-relation between decreasing cytokinin levels and increasing cytokinin oxidase activity in developing maize kernels (Dietrich et al., 1995). One function of cytokinin oxidase might be to destroy endogenous cytokinins at the appropriate time during kernel development (Bilyeu ata 1., 2001). An alternative role for cytokinin oxidase activity may be the protection of the kernel from pathogen invasion, because fungal pathogens are known to be capable of producing cytokinins (Mills & Van Staden, 1978; Angra et al., 1990).
In order to test the hypothesis that cytokinin levels in the root indeed exceed the level optimal for root growth, novel genes encoding cytokinin oxidases (which are cytokinin metabolising enzymes) were cloned from Arabidopsis thaliana (designated AtCKX) and were subsequently expressed under a strong constitutive promoter in transgenic tobacco and Arabidopsis. Transformants showing AtCKX mRNA expression and increased cytokinin oxidase activity also manifested enhanced formation and growth of roots. Negative effects on shoot growth were also observed. The latter is in accordance with the constitutive expression of the cytokinin oxidase gene in these plants, illustrating the importance of confined expression of the cytokinin oxidase gene for general plant growth properties. Containment of cytokinin oxidase activity can be achieved by using cell-, tissue- or organ-specific promoters, since cytokinin degradation is a process limited to the tissues or cells that express the CKX protein, this in contrast to approaches relying on hormone synthesis, as explained above.
The observed positive effects of cytokinin oxidase expression on root growth demonstrate that cytokinin oxidases are interesting targets for the design of or screening for herbicides. Such herbicides should inhibit cytokinin oxidase activity, should preferably not be transported to the shoot, and should be soluble and relatively stable in a solvent that can be administered to the root through the soil.
In accordance with the present invention, it has also been surprisingly discovered that transgenic plants overexpressing a cytokinin oxidase gene develop seeds (including embryos) and cotyledons of increased size and/or weight. These results are surprising as reduced cytokinin content would have been expected to be associated with a reduced organ growth.

Numerous reports ascribe a stimulatory or inhibitory function to cytokinins in different developmental processes such as root growth and branching, control of apical dominance in the shoot, chloroplast development, and leaf senescence (Mok M.C. (1994) in Cytokines: Chemistry, Activity and Function, eds., Mok, D. W. S. & Mok, M. C. (CRC Boca Raton, Fla.), pp.155-166). Conclusions about the biological functions of cytokinins have mainly been derived from studies on the consequences of exogenous cytokinin application or endogenously enhanced cytokinin levels (Klee, H. J. & Lanehon, M. B. (1995) in Plant Hormones:Physiology, Biochemisry and Molecular Biology, ed. Davies, P. J. (Kluwer, Dordrdrocht, the Netherlands), pp. 340-353, Smulling, T., Rupp, H. M. Frank, M & Schafer, S. (1999) in Advances in Regulation of Plant Growth and Development, eds. Surnad, M. Pac P. & Beck, E. (Peres, Prague), pp. 85-96). Up to now, it has not been possible to address the reverse question: what are the consequences for plant growth and development if the endogenous cytokinin concentration is decreased? Plants with a reduced cytokinin content are expected to yield more precise information about processes cytokinins limit and, therefore, might regulate. Unlike other plant hormones such as abscisic acid, gibberellins, and ethylene, no cytokinin biosynthetic mutants have been isolated (Hooykens, P. J. J., Hall, M. A. & Libbeuga, K. R., eds.(1999) Biochemistry and Molecular Biology of Plant Hormones (Elsevier, Amsterdam).
The catabolic enzyme cytokinin oxidase (CKX) plays a principal role in controlling cytokinin levels in plant tissues. CKX activity has been found in a great number of higher plants and in different plant tissues. The enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins bearing unsaturated isoprenoid side chains. The free bases iP and Z, and their respective ribosides are the preferred substrates. The reaction products of iP catabolism are adenine and the unsaturated aldehyde 3-methyl-2-butonal (Armstrong, D. J. (1994) in Cytokinins: Chemistry, Activity and Functions, eds. Mok. D. W. S & Mok, M. C. (CRC Boca Raton, Fla.), pp. 139-154). Recently, a cytokinin oxidase gene from Zea mays has been isolated (Morris, R. O., Bilyeu, K. D., Laskey, J. G. & Cherich, N. N. (1999) Biochem. Biophys.Res. Commun. 255, 328-333, Houba-Heria, N.,Pethe, C. d'Alayer, J & Lelouc, M. (1999) Plant J. 17:615-626). The manipulation of CKX gene expression could partially overcome the lack of cytokinin biosynthetic mutants and can be used as a powerful tool to study the relevance of iP- and Z-type cytokinins during the whole life cycle of higher plants.
1. mRNA purification was performed by first, isolating high quality total RNA from 6 day old RASI seedlings and, subsequently by isolating mRNA from total RNA using oligo (dT) cellulose in a filter syringe by making use of a double purification method.
2. mRNA was converted into first and second strand cDNA followed by Sal I adapter addition, Not I digestion, cDNA vector ligation and transformation to obtain the cDNA library.
3. The superscript plasmid system with Gateway for cDNA cloning and synthesis was employed throughout.
4. The clones obtained were picked, digested using Not I and Sal I enzymes, to obtain the inserts and these were further sequenced and checked for homology.
5. The sequencing of the selected clones was done on ABI Prism, 377, DNA Sequencer (Perkin Elmer).

In biotechnology, cytokinins are important because they control regeneration of whole plants from single cells and the clonal propagation of unique genetic lines. The genetic and biochemical characterisation of the cytokinin oxidase gene will provide an insight into its role in regulating the reversible and irreversible inactivation of cytokinins in plants.

We claim
1. A method of targeted manipulation of cytokinin oxidase gene expression
serves as an important and novel tool to modulate growth characteristics and
yield parameters of crop plants via a differential influence on the number
and/or duration of cell division cycles in the root and shoot meristems.
2. A claim as in claim 1, wherein the transgenic plants could apply to all
varieties of plants.
3. A claim as in claim 1 & 2, wherein, the transgenic plants expressing the
cytokinin oxidase gene, confer maintenance of an optimal level of cytokinins
for growth and/or resetting a cytokinin signaling system to a basal level.
4. A claim as in claim 2, wherein the transgenic plants having reduced levels of
cytokinins, display a stimulatory or inhibitory effect on different
developmental processes such as root growth and branching, control of
apical dominance in the shoot, chloroplast development, and leaf senescence


733-che-2003 abstract 28-07-2009.pdf

733-che-2003 claims 28-07-2009.pdf

733-che-2003 correspondence others 28-07-2009.pdf

733-che-2003 description(complete) 28-07-2009.pdf

733-che-2003 drawings 28-07-2009.pdf

733-che-2003 form-1 28-07-2009.pdf




733-che-2003-form 1.pdf

744-che-2003 others- 22-07-2009.pdf

744-che-2003 claims 22-07-2009.pdf

Patent Number 241758
Indian Patent Application Number 733/CHE/2003
PG Journal Number 31/2010
Publication Date 30-Jul-2010
Grant Date 23-Jul-2010
Date of Filing 16-Sep-2003
Name of Patentee Avestha Gengraine Technologies Pvt Ltd
# Inventor's Name Inventor's Address
PCT International Classification Number C12N15/82
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA