Protein involved in abscisic acid signalling

Details Protein involved in abscisic acid signalling Protein involved in abscisic acid signalling

2000-05-24

2004-11-23

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C530S350000, C530S300000, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000, C530S330000

Reexamination Certificate

active

06821735

ABSTRACT:

The present invention relates to an absicisic acid (ABA) signalling component, nucleic acid encoding the signalling component, and processes employing the same.
The plant phytohormone ABA plays an important role in growth and development of plants. In seeds it helps in embryogenesis and formation of seed-storage proteins. It prevents premature germination and growth of many seeds and buds. In vegetative tissue ABA protects the plant against adverse environmental conditions such as drought, high salinity, cold temperature or frost. Many of the actions of ABA result in rather long-term physiological changes and appear mainly to involve modification of gene expression at transcriptional level. Over 150 ABA responsive genes have been isolated from various plant species.
The importance of ABA as a stress hormone in connection with a lack of water was first suggested in 1969 by STC Wright and RWP Hiron at Wye College, London University. They found that ABA content of wheat leaves rose by a factor of 40 during the first half hour of wilting. Similar rise in ABA is now detected in leaves of monocot as well as dicot plants. Application of ABA to leaves causes stomatal closing or inhibits stomatal opening as observed in numerous species. Therefore, during water stress, increased ABA-levels reduce water loss.
We have found a novel protein that mediates in signalling evoked by ABA, and which responds to ABA.
According to the present invention there is now provided a protein capable of affecting an ABA response and comprising one or more of the following:
(i) a hydrophobic C-terminus;
(ii) at least one coiled coil region;
(iii) an EF-hand consensus sequence;
(iv) a nucleotide binding site; and
(v) a hydrophilic N-terminus;
or a variant thereof.
In a preferred embodiment, the protein has (i), (ii), (iii) and (iv) as defined above, and optionally (v).
Further, the protein may also be capable of being cleaved by botulinum C. Thus preferably the protein includes two recognition domains for botulinum C and/or a cleavage site.
Preferably at least one of the coiled coil regions corresponds to an epimorphin pattern.
Preferably there are three coiled coil regions.
Preferably there are three coiled coil regions, at least one of which corresponds to an epimorphin pattern.
Preferably the protein further comprises phosphorylation sites.
The protein may be described as a novel syntaxin (t-SNARE) homolog. At the cellular level, ABA is best characterised by its action in regulating K
+
ands Cl
−
channels at the plasma membrane of stomatal guard cells, leading to stomatal closure that reduces transpirational water loss from leaf. The protein of the present invention may be a membrane-anchored protein that is associated with the plasma membrane. In vivo, both cleavage of the protein by Botulinum C toxin and competition by a soluble C-truncated fragment of the protein have been found to prevent ABA action in controlling K
+
and Cl
−
channels in the guard cells. These, and additional results, show that the protein of the present invention is involved in an ABA signalling complex, and responds to ABA.
The features of the protein can be defined as follows with reference to SEQ ID NO:24
Feature
Intervals — amino acids
(i)
hydrophobic C-terminus
282-296/280-294
(ii)
coiled coil region/epimorphin pattern
210-247/216-240
(iii)
an EF-hand consensus sequence
16-28
(iv)
nucleotide binding site
116, 118 and 120/114-119.
(v)
hydrophilic N-terminus
1-281/1-279
(vi)
botulinum cleavage sites
269-274
In an especially preferred embodiment, the protein comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4.
The present invention includes variants of the protein defined above. Such variants include proteins having 50% or more overall homology with the sequence of SEQ ID NO:2. Typically the homology is 60% or more, more typically 65%, preferably 70%, more preferably 75%, even more preferably 80% or 85%, especially preferred are 90%, 95%, 98% or 99% homology.
Percentage homology preferably is calculated on the basis of amino acids that are identical in corresponding positions in the two sequences under consideration. Conservative substitutions are not taken into account. In calculation of percentage homology of a putative protein under investigation with the SEQ ID NO:2 or SEQ ID NO:4, if the protein under investigation has a different length, then the calculation is based on the amino acids in the portion of the molecule under investigation that overlaps with the sequence shown in SEQ ID NO:2 or SEQ ID NO: 4.
In particular, the term “homology” as used herein may be equated with the term “identity”.
Here, sequence homology can be determined by a simple “eyeball” comparison of any one or more of the sequences with another sequence to see if that other sequence has at least 75% identity to the sequence(s).
Relative sequence homology (i.e. sequence identity) can also be determined by commercially available computer programs that can calculate % homology between two or more sequences. A typical example of such a computer program is CLUSTAL.
Sequence homology (or identity) may moreover be determined using any suitable homology algorithm, using for example default parameters. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail in, for example: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410; Madden, T. L., Tatusov, R. L. & Zhang, J. (1996) “Applications of network BLAST server” Meth. Enzymol. 266:131-141.; Gish, W. & States, D. J. (1993) “Identification of protein coding regions by database similarity search.” Nature Genet. 3:266-272; Altschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402; Karlin, S. & Altschul, S. F. (1990) “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes.” Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S. & Altschul, S. F. (1993) “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc. Natl. Acad. Sci. USA 90:5873-5877, which are incorporated herein by reference. The search parameters are defined as follows, and are advantageously set to the defined default parameters.
Advantageously, “substantial homology” when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul with a few enhancements.
Analysis may also be carried out using Lasergene DNA, Madison, U.S.A.
It will be appreciated that proteins capable of affecting an ABA response and/or responding to ABA from other species will exhibit inter-species differences, for example, differences in protein length, amino acid sequence and carbohydrate modifications. There may, for example, be variations in the C- and/or N-terminal residues, and in molecular weight.
In a general sense the term “variant” includes a protein which retains the essential properties, in the present case the ability to affect an ABA response and/or respond to ABA. Variants include allelic variants, and proteins, which differ by conservative amino acid changes. The variants may be natural or non-naturally occurring variants made, for example, by mutagenesis.
By conservative amino acid changes we mean replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methio

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