TECHNICAL FIELD
The present invention relates methods for producing monocotyledonous plants
from the Poaceae family with at least one of: increased root biomass and
increased above-ground biomass.
BACKGROUND ART
The Poaceae (also called Gramineae or true grasses) family of monocotyledonous
plants is the most economically important plant family in modern times, providing
numerous human food crops, and also species useful for forage, building
materials (bamboo, thatch) and biofuel production.
Some of these applications are limited in part at least, by the plant's architecture
and productivity, including the amount of root biomass and above-ground
biomass produced.
Poaceae plants with increased above-ground biomass would have a number of
advantages, particularly for crops where above-ground parts of the plant are
harvested, in biofuel crops, and in forage crops.
Poaceae plants with increased root biomass would potentially have a number of
advantages including better anchorage, more efficient water uptake, more
efficient nutrient uptake, and improved drought tolerance. A combination of
these features may also result in improved yield, including grain and leaf
biomass.
At present there is limited understanding of the genetic mechanisms controlling
production of root and above-ground biomass in Poaceae plants.
It would therefore be beneficial to have available alternative methods for
controlling root and above-ground biomass in Poaceae plants.
It is therefore an object of the invention to provide methods and materials for
altering the production of at least one of root biomass and above-ground biomass
in Poaceae plants, and/or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
Previously, White (2006) discovered two adjacent homologous genes in
Arabidopsis (named PEAPOD, PPD1 and PPD2) that reg ulate the cel l prol iferation
of meristemoids duri ng the late stages of leaf and seed pod development.
Homologs of these genes were fou nd in mosses, all dicotyledonous plants,
con ifers and palms but were fou nd to be absent from the grass fam ily (Poaceae) .
Deletion of these genes in Arabidopsis resulted in enlarged leaves and wide seed
pods while over expression of PPD 1 resu lted in a reduction in the size of the
leaves and siliq ues (Wh ite, 2006) . In add ition a red uction in PPD expression
com bined with over expression of either the brassi nosteroid receptor (BRI 1) or a
member of the auxi n responsive gene fam ily (SAUR19) demonstrated positive
epistasis with respect to leaf growth in Arabidopsis (Vanhaeren et al 2014) .
The applicants have now surprisi ngly shown that the expression of PEAPOD
protei ns in Poaceae plants resu lts in an increase in the prod uction of root and
above-g round biomass.
The appl icant's invention therefore relates to a method for increasi ng at least one
of root biomass and above-g rou nd biomass in Poaceae plants by ectopic
expression of PEAPOD. In particu lar the invention relates to expressi ng PEAPOD
protei ns that are characterized by presence of at least one consensus ami no acid
motif com mon to all PEAPOD protei ns disclosed from a wide range of plant
species.
Because Poaceae plants do not natural ly contai n PEAPOD genes, the plants used
in, or prod uced by the methods of the invention do not occur in natu re.
Methods
In the f irst aspect the invention provides a method for increasi ng at least one of
root biomass and above-g round biomass and in a Poaceae plant, the method
com prisi ng the step of expressi ng a PEAPOD protei n in the Poaceae plant.
In one embod iment at least one of root biomass and above-g rou nd biomass is
increased relative to that in a control plant, of the same species or variety, which
does not express the PEAPOD protei n.
In one embodiment the PEAPOD protein is expressed as a consequence of the
plant, or its ancestor plant or plant cell having been transformed with a
polynucleotide encoding the PEAPOD protein.
In a further embodiment, the plant is transgenic for a polynucleotide expressing
the PEAPOD protein.
In a further aspect the invention provides a method for producing a Poaceae plant
with at least one of increased root biomass and increased above-ground biomass,
the method comprising the step of expressing a PEAPOD protein in the Poaceae
plant.
In one embodiment the Poaceae plant is transformed with a polynucleotide
encoding the PEAPOD protein.
In a further embodiment the method comprises the step of transforming the
Poaceae plant, or transforming a Poaceae plant cell which is regenerated into the
Poaceae plant, with a polynucleotide encoding the PEAPOD protein.
In one embodiment the method includes the additional step of testing or
assessing the plant for at least one of increased root biomass and increased
above-ground biomass. In one embodiment the method includes the additional
step of testing or assessing the plant for increased above-ground biomass. In
one embodiment the method includes the additional step of testing or assessing
the plant for increased root biomass.
In a further embodiment the method includes the step producing further plants
with at least one of increased root biomass and increased above-ground biomass,
by asexually or sexually multiplying the plants tested for at least one of increased
root biomass and increased above-ground biomass.
PEAPOD proteins
In one embodiment the PEAPOD protein is a polypeptide comprising the sequence
of at least one of SEQ ID NO: 28, 29, 31, 32, 34 and 35.
In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID
NO: 28. In a further embodiment the PEAPOD protein comprises the sequence of
SEQ ID NO: 29. In a further embodiment the PEAPOD protein comprises the
sequence of SEQ ID NO: 31. In a further embodiment the PEAPOD protein
comprises the sequence of SEQ ID NO: 32. In a further embodiment the PEAPOD
protein comprises the sequence of SEQ ID NO: 34. In a further embodiment the
PEAPOD protein comprises the sequence of SEQ ID NO:35.
In a further embodiment the PEAPOD protein is a polypeptide comprising a
sequence with at least 70% identity to any one of SEQ ID NO: 1 to 26.
In a further embodiment the PEAPOD protein is a polypeptide comprising a
sequence selected from any one of SEQ ID NO: 1 to 26.
In a further embodiment the PEAPOD protein is a polypeptide comprising a
sequence with at least 70% identity to SEQ ID NO: 1.
In a further embodiment the PEAPOD protein is a polypeptide comprising the
sequence of SEQ ID NO: 1.
Expressing PEAPOD
Methods for expressing proteins in plants are well known to those skilled in the
art, and are described herein. All of such methods are included within the scope
of the invention.
Increasing expression of PEAPOD by introducing a polynucleotide
In one embodiment expression is increased by introducing a polynucleotide into
the plant cell or plant.
In a preferred embodiment the polynucleotide encodes a PEAPOD protein as
herein defined.
In a further embodiment the polynucleotide comprises a sequence with at least
70% identity to the coding sequence of any one of SEQ ID NO: 80 to 104.
In a further embodiment the polynucleotide comprises a sequence with at least
70% identity to the sequence of any one of SEQ ID NO: 80 to 104.
In a further embodiment the polynucleotide comprises the coding sequence of
any one of SEQ ID NO: 80 to 104.
In a further embodiment the polynucleotide comprises the sequence of any one of
SEQ ID NO: 80 to 104.
In a further embodiment the polynucleotide comprises a fragment of the
sequences described above, that is capable of encoding a polypeptide with the
same function as a PEAPOD protein. In one embodiment the fragment encodes a
polypeptide capable of increasing at least one of leaf and root biomass.
Expressing PEAPOD via an expression construct
In a preferred embodiment the polynucleotide is introduced into the plant as part
of an expression construct.
In a preferred embodiment the expression construct comprises a promoter
operatively linked to the polynucleotide.
Promoter for increasing expression of PEAPOD
In one embodiment the promoter is capable of driving, or drives, expression of
the operatively linked polynucleotide constitutively in all tissues of the plant.
In a further embodiment the promoter is a tissue-preferred promoter.
In a further embodiment the promoter is capable of driving, or drives, expression
of the operatively linked polynucleotide in the above-ground parts of the plant.
In a further embodiment the promoter is capable of driving, or drives, expression
of the operatively linked polynucleotide in the leaves of the plant.
In one embodiment the promoter is an above-ground parts- preferred promoter.
In one embodiment the promoter is a leaf-preferred promoter.
In a further embodiment the promoter is a leaf specific promoter.
In a further embodiment the promoter is capable of driving, or drives, expression
of the operatively linked polynucleotide in the below ground tissues of the plant.
In one embodiment the promoter is a below ground tissues-preferred promoter.
In a further embodiment the promoter is a below ground tissue-specific promoter.
In one embodiment the promoter is a light-repressed promoter.
In a further embodiment the promoter is capable of driving, or drives, expression
of the operatively linked polynucleotide in the roots of the plant.
In one embodiment the promoter is a root- preferred promoter.
In a further embodiment the promoter is a root-specific promoter.
Source of polynucleotides and polypeptides
The polynucleotides and variants of polynucleotides of the invention, or used in
the methods of the invention, may be derived from any species. The
polynucleotides and variants may also be synthetically or recombinantly
produced, and also may be the products of "gene shuffling" approaches.
The polypeptides and variants of polypeptides of the invention, or used in the
methods of the invention, may be derived from any species. The polypeptides
and variants may also be recombinantly produced and also may also be
expressed from the products of "gene shuffling' approaches.
In one embodiment the polynucleotide, polypeptide or variant, is derived from a
plant species.
In a further embodiment the polynucleotide, polypeptide or variant, is derived
from gymnosperm plant species.
In a further embodiment the polynucleotide, polypeptide or variant, is derived
from an angiosperm plant species.
In a further embodiment the polynucleotide, polypeptide or variant, is derived
from a dicotyledonous species.
In a preferred embodiment the polynucleotide, polypeptide or variant, is derived
from a eudicot species.
In a further embodiment the polynucleotide, polypeptide or variant, is derived
from a monocotyledonous species. Preferred monocot plants include: palm,
banana, duckweed and orchid species.
Poaceae plant cells and plants to be transformed
Preferred Poaceae subfamilies include the: Anomochlooideae, Pharoideae,
Puelioideae, Bambusoideae, Pooideae, Ehrhartoideae, Aristidoideae,
Arundinoideae, Chloridoideae, Panicoideae, Danthonioideae, and Micrairoideae.
A preferred Poaceae family is the subfamily pooideae. Preferred pooideae plants
include wheat, barley, oats, brome grass and reed grass.
Another preferred Poaceae family is the subfamily ehrhartoideae. Preferred
ehrhartoideae plants include rice.
Another preferred Poaceae family is the subfamily panicoideae. Preferred
panicoideae plants include panic grass, maize, sorghum, sugar cane, energy
cane, millet, fonio and bluestem grasses.
Another preferred Poaceae family is the subfamily Arundinoideae. Preferred
Arundinoideae plants include Arundo donax.
Another preferred Poaceae family is the subfamily Bambusoideae. Preferred
Bambusoideae plants include bamboo.
Preferred Poacea species include those form the Lolium genera. Preferred Lolium
species include Lolium longiflorum, Lolium multiflorum, Lolium perenne, Lolium
westerwoldicum, Lolium temulentum, and Lolium hybridum.
Other preferred Poacea species include those form the Festuca genera. Preferred
Festuca species include Festuca arundinacea, Festuca ovina, Festuca pratensis
and Festuca rubra.
Plants and plant parts
In a further aspect the invention provides a Poaceae plant expressing a PEAPOD
protein, or fragment thereof, that has at least one of:
a) increased root biomass, and
b) increased above-ground biomass,
as a result of expressing the PEAPOD protein, or fragment thereof.
In one embodiment the PEAPOD protein, or fragment thereof, is expressed as a
consequence of the plant, or its ancestor plant or plant cell, having been
transformed with a polynucleotide encoding the PEAPOD protein, or fragment
thereof.
In a further embodiment the Poaceae plant is transgenic for a polynucleotide
expressing the PEAPOD protein, or fragment thereof.
In a further embodiment the polynucleotide or fragment thereof is operatively
linked polynucleotide to a tissue-preferred promoter.
In one embodiment the promoter is capable of driving, or drives, expression of
the operatively linked polynucleotide, or a fragment thereof, in the above-ground
parts of the plant.
In a further embodiment the promoter is capable of driving, or drives, expression
of the operatively linked polynucleotide, or a fragment thereof, in the below
ground tissues of the plant.
In a further embodiment the PEAPOD protein is as herein defined.
In a further embodiment the polynucleotide, encoding the PEAPOD protein, is as
herein defined.
In a further embodiment the Poaceae plant is as herein defined.
In a further aspect the invention provides a cell, part, propagule or progeny of
the plant that is transgenic for at least one of:
a) the polynucleotide, and
b) the polynucleotide and operatively linked promoter.
DETAILED DESCRIPTION
In this specification where reference has been made to patent specifications,
other external documents, or other sources of information, this is generally for
the purpose of providing a context for discussing the features of the invention.
Unless specifically stated otherwise, reference to such external documents is not
to be construed as an admission that such documents, or such sources of
information, in any jurisdiction, are prior art, or form part of the common general
knowledge in the art.
The term "comprising" as used in this specification means "consisting at least in
part of". When interpreting each statement in this specification that includes the
term "comprising", features other than that or those prefaced by the term may
also be present. Related terms such as "comprise" and "comprises" are to be
interpreted in the same manner.
Increased root biomass
A plant with "increased root biomass" produces more root biomass than does a
control plant of the same type and age. Thus "increased" means increased
relative to a control plant of the same type and age.
Preferably the plant with "increased root biomass" produces at least 10%,
preferably at least 20%, more preferably at least 30%, more preferably at least
40%, more preferably at least 50%, more preferably at least 60%, more
preferably at least 70%, more preferably at least 80%, more preferably at least
90%, more preferably at least 100%, more preferably at least 150%, more
preferably at least 200%, more preferably at least 300%, more preferably at
least 400% more root biomass than does a control plant of the same type and
age.
In one embodiment the plant with "increased root biomass" has at least one of:
larger roots, longer roots, more roots, more lateral roots, or a more extensive
root system, than does a control plant.
Root biomass
The term root biomass refers to total mass of root tissue produced by the plant.
This can be assessed by dry weight or wet weight.
Root
The term root as used herein encompasses the primary root, secondary roots,
adventitious roots, root branches and root hairs. Roots are generally below
ground, but the term also encompasses aerial roots. In one embodiment the
term root encompasses non-leaf, non-node bearing parts of the plant.
Increased drought tolerance
In one embodiment the plant with "increased root biomass" also has increased
drought tolerance. Again "increased" means increased relative to a control plant
of the same type and age.
The term "increased drought tolerance" is intended to describe a plant, or plants,
which perform more favourably in any aspect of their growth and development
under sub-optimal hydration conditions than do suitable control plants in the
same conditions.
Increased above-ground biomass
A plant with "increased above-ground biomass" produces more above-ground
biomass than does a control plant of the same type and age. Thus "increased"
means increased relative to a control plant of the same type and age.
Preferably the plant with "increased above-ground biomass" produces at least
10%, preferably at least 20%, more preferably at least 30%, more preferably at
least 40%, more preferably at least 50%, more preferably at least 60%, more
preferably at least 70%, more preferably at least 80%, more preferably at least
90%, more preferably at least 100%, more preferably at least 150%, more
preferably at least 200%, more preferably at least 300%, more preferably at
least 400% more above-ground biomass than does a control plant of the same
type and age.
In one embodiment the plant with "increased above-ground biomass" has at least
one of: larger leaves, more leaves, a longer stem (culm), a thicker stem (culm),
more tillers, larger tillers, more stolons, larger stolons than does a control plant.
Preferably the plant with "increased above-ground biomass" has larger leaves
than does a control plant.
Above-ground biomass
The term above-ground biomass refers to total mass of above-ground tissue
produced by the plant. This can be assessed by dry weight or wet weight.
Above ground biomass can be contributed to by any one of leaves,
stems/culms/tillers/and stolons.
Leaf
The term leaf as used herein means the same as standard usage of the term.
Preferably the term leaf includes the leaf blade (or leaf lamina) and any leaf stalk.
Stem/culm
The stem (or culm) is the central axis of the mature grass shoot, comprised of
nodes and internodes, each node bearing a leaf.
Tiller
A tiller is a daughter plant, a shoot capable of producing a new plant.
Stolon
A stolon is a prostrate or creeping, above-ground stem, rooting at the nodes, and
is a means of vegetative reproduction.
Increased flower branching
In one embodiment the plant with at least one of increased root biomass and
increased above-ground biomass" also has "increased flower branching". Again
"increased" means increased relative to a control plant of the same type and age.
The term "increased flower branching" means at least one of: an increase in the
number of stalks bearing inflorescences, and an increase in the number of
spikelets within an inflorescence.
Increased seed yield
In one embodiment the plant with "increased flower branching" also has
"increased seed yield". Again "increased" means increased relative to a control
plant of the same type and age.
A plant with "increased seed yield" produces more seed biomass than a control
plant of the same type and age. This can be assessed by dry weight or wet
weight. A plant with increased seed yield may produce more seeds, and/or larger
seeds than a control plant. Preferably, the plant produced more seed than a
control plant.
Control plant
In one embodiment the control plant is a wild-type plant. In a further
embodiment the control plant is a plant that does not express a PEAPOD gene. In
a further embodiment the control plant is a non-transformed plant. In a further
embodiment the control plant is a plant that has not been transformed with a
PEAPOD polynucleotide. In a further embodiment the control plant is a plant that
has not been transformed with a construct. In a further embodiment the control
plant is a plant that has been transformed with a control construct. In one
embodiment the construct is an empty vector construct.
Tissue preferred promoters
In certain embodiments, the PEAPOD protein encoding polynucleotides are
expressed under the control of tissue preferred promoters. The term "preferred"
with respect to tissue preferred promoters means that the promoter primarily
drives expression in that tissue. Thus, for example, a leaf-preferred promoter
drives a higher level of expression of an operably linked polynucleotide in leaf
tissue than it does in other tissues or organs or the plant. Similarly a rootpreferred
promoter drives a higher level of expression of an operably linked
polynucleotide in root tissue than it does in other tissues or organs or the plant.
Leaf-preferred promoters
A leaf-preferred promoter drives a higher level of expression of an operably linked
polynucleotide in leaf tissue than it does in other tissues or organs or the plant.
Leaf preferred promoters may include photosynthetic tissue preferred promoters
and light regulated promoters.
Photosynthetic tissue preferred promoters
Photosynthetic tissue preferred promoters include those that are preferentially
expressed in photosynthetic tissues of the plants. Photosynthetic tissues of the
plant include leaves, stems, shoots and above ground parts of the plant.
Photosynthetic tissue preferred promoters include light regulated promoters.
Light regulated promoters
Numerous light regulated promoters are known to those skilled in the art and
include for example chlorophyll a/b (Cab) binding protein promoters and Rubisco
Small Subunit (SSU) promoters. An example of a light regulated promoter is
found in US 5,750,385. Light regulated in this context means light inducible or
light induced.
Root preferred promoters
A root-preferred promoter drives a higher level of expression of an operably
linked polynucleotide in root tissue than it does in other tissues or organs or the
plant.
Root-preferred promoters may include non-photosynthetic tissue preferred
promoters and light-repressed regulated promoters.
Non-photosynthetic tissue preferred promoters
Non-photosynthetic tissue preferred promoters include those preferentially
expressed in non-photosynthetic tissues/organs of the plant.
Non-photosynthetic tissue preferred promoters may also include light repressed
promoters.
Light repressed promoters
An example of a light repressed promoter is found in US 5,639,952 and in US
5,656,496.
Root specific promoters
An example of a root specific promoter is found in US 5,837,848; and US
2004/0067506 and US 2001/0047525.
The term "preferentially expressed" with respect to a promoter being
preferentially expressed in a certain tissue, means that the promoter is expressed
at a higher level in that tissue than in other tissues of the plant.
The term "tissue specific" with respect to a promoter, means that the promoter is
expressed substantially only in that tissue, and not other tissues of the plant.
In one embodiment the leaf-preferred promoter is a leaf-specific promoter.
In one embodiment the root- preferred promoter is a root-specific promoter.
The term "gene" as used herein means an endogenous genomic sequence which
includes a coding sequence which encodes a polypeptide or protein. The coding
sequence may be interrupted by one or more introns. A gene typically also
includes a promoter sequence, 5' untranslated sequence, 3' untranslated
sequence, and a terminator sequence. Genomic sequences that regulate
expression of the protein may also be considered part of the gene.
Polynucleotides and fragments
The term "polynucleotide(s)," as used herein, means a single or double-stranded
deoxyhbonucleotide or ribonucleotide polymer of any length but preferably at
least 15 nucleotides, and include as non-limiting examples, coding and noncoding
sequences of a gene, sense and antisense sequences complements, exons,
introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA,
ribozymes, recombinant polypeptides, isolated and purified naturally occurring
DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes,
primers and fragments.
A "fragment" of a polynucleotide refers to a contiguous subsequence of larger a
polynucleotide sequence. Preferably the fragment is at least 15 nucleotides
preferably at least 16 nucleotides, more preferably at least 17 nucleotides, more
preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more
preferably at least 20 nucleotides, more preferably at least 21 nucleotides, more
preferably at least 22 nucleotides, more preferably at least 23 nucleotides, more
preferably at least 24 nucleotides, more preferably at least 25 nucleotides, more
preferably at least 26 nucleotides, more preferably at least 27 nucleotides, more
preferably at least 28 nucleotides, more preferably at least 29 nucleotides, more
preferably at least 30 nucleotides, more preferably at least 31 nucleotides, more
preferably at least 32 nucleotides, more preferably at least 33 nucleotides, more
preferably at least 34 nucleotides, more preferably at least 35 nucleotides, more
preferably at least 36 nucleotides, more preferably at least 37 nucleotides, more
preferably at least 38 nucleotides, more preferably at least 39 nucleotides, more
preferably at least 40 nucleotides, more preferably at least 4 1 nucleotides, more
preferably at least 42 nucleotides, more preferably at least 43 nucleotides, more
preferably at least 44 nucleotides, more preferably at least 45 nucleotides, more
preferably at least 46 nucleotides, more preferably at least 47 nucleotides, more
preferably at least 48 nucleotides, more preferably at least 49 nucleotides, more
preferably at least 50 nucleotides, more preferably at least 51 nucleotides, more
preferably at least 52 nucleotides, more preferably at least 53 nucleotides, more
preferably at least 54 nucleotides, more preferably at least 55 nucleotides, more
preferably at least 56 nucleotides, more preferably at least 57 nucleotides, more
preferably at least 58 nucleotides, more preferably at least 59 nucleotides, more
preferably at least 60 nucleotides, more preferably at least 6 1 nucleotides, more
preferably at least 62 nucleotides, more preferably at least 63 nucleotides, more
preferably at least 64 nucleotides, more preferably at least 65 nucleotides, more
preferably at least 66 nucleotides, more preferably at least 67 nucleotides, more
preferably at least 68 nucleotides, more preferably at least 69 nucleotides, more
preferably at least 70 nucleotides, more preferably at least 7 1 nucleotides, more
preferably at least 72 nucleotides, more preferably at least 73 nucleotides, more
preferably at least 74 nucleotides, more preferably at least 75 nucleotides, more
preferably at least 76 nucleotides, more preferably at least 77 nucleotides, more
preferably at least 78 nucleotides, more preferably at least 79 nucleotides, more
preferably at least 80 nucleotides, more preferably at least 8 1 nucleotides, more
preferably at least 82 nucleotides, more preferably at least 83 nucleotides, more
preferably at least 84 nucleotides, more preferably at least 85 nucleotides, more
preferably at least 86 nucleotides, more preferably at least 87 nucleotides, more
preferably at least 88 nucleotides, more preferably at least 89 nucleotides, more
preferably at least 90 nucleotides, more preferably at least 9 1 nucleotides, more
preferably at least 92 nucleotides, more preferably at least 93 nucleotides, more
preferably at least 94 nucleotides, more preferably at least 95 nucleotides, more
preferably at least 96 nucleotides, more preferably at least 97 nucleotides, more
preferably at least 98 nucleotides, more preferably at least 99 nucleotides, more
preferably at least 100 nucleotides, more preferably at least 150 nucleotides,
more preferably at least 200 nucleotides, more preferably at least 250
nucleotides, more preferably at least 300 nucleotides, more preferably at least
350 nucleotides, more preferably at least 400 nucleotides, more preferably at
least 450 nucleotides and most preferably at least 500 nucleotides of contiguous
nucleotides of a polynucleotide disclosed. A fragment of a polynucleotide
sequence can be used in antisense, RNA interference (RNAi), gene silencing,
triple helix or ribozyme technology, or as a primer, a probe, included in a
microarray, or used in polynucleotide-based selection methods of the invention.
In one embodiment the fragment encodes a polypeptide that performs, or is
capable of performing, the same function as the polypeptide encoded by the
larger polynucleotide that the fragment is part of.
The term "primer" refers to a short polynucleotide, usually having a free 3Ό H
group that is, or can be, hybridized to a template and used for priming
polymerization of a polynucleotide complementary to the target.
The term "probe" refers to a short polynucleotide that is, or can be, used to
detect a polynucleotide sequence that is complementary to the probe, in a
hybridization-based assay. The probe may consist of a "fragment" of a
polynucleotide as defined herein.
Polypeptides and fragments
The term "polypeptide", as used herein, encompasses amino acid chains of any
length but preferably at least 5 amino acids, including full-length proteins, in
which amino acid residues are linked by covalent peptide bonds. Polypeptides of
the present invention, or used in the methods of the invention, may be purified
natural products, or may be produced partially or wholly using recombinant or
synthetic techniques. The term may refer to a polypeptide, an aggregate of a
polypeptide such as a dimer or other multimer, a fusion polypeptide, a
polypeptide fragment, a polypeptide variant, or derivative thereof.
A "fragment" of a polypeptide refers to a contiguous subsequence of larger a
polypeptide. Preferably the fragment is at least 5, more preferably at least 10,
more preferably at least 20, more preferably at least 30, more preferably at
least 40, more preferably at least 50, more preferably at least 100, more
preferably at least 120, more preferably at least 150, more preferably at least
200, more preferably at least 250, more preferably at least 300, more
preferably at least 300 , more preferably at least 400 amino acids in length.
In one embodiment the fragment performs, or is capable of performing, the same
function as the polypeptide that the fragment is part of.
Preferably the fragment performs a function that is required for the biological
activity and/or provides three dimensional structure of the polypeptide.
The term "isolated" as applied to the polynucleotide or polypeptide sequences
disclosed herein is used to refer to sequences that are removed from their
natural cellular environment. In one embodiment the sequence is separated
from its flanking sequences as found in nature. An isolated molecule may be
obtained by any method or combination of methods including biochemical,
recombinant, and synthetic techniques.
The term "recombinant" refers to a polynucleotide sequence that is synthetically
produced or is removed from sequences that surround it in its natural context.
The recombinant sequence may be recombined with sequences that are not
present in its natural context.
A "recombinant" polypeptide sequence is produced by translation from a
"recombinant" polynucleotide sequence.
The term "derived from" with respect to polynucleotides or polypeptides of the
invention being derived from a particular genera or species, means that the
polynucleotide or polypeptide has the same sequence as a polynucleotide or
polypeptide found naturally in that genera or species. The polynucleotide or
polypeptide, derived from a particular genera or species, may therefore be
produced synthetically or recombinantly.
Variants
As used herein, the term "variant" refers to polynucleotide or polypeptide
sequences different from the specifically identified sequences, wherein one or
more nucleotides or amino acid residues is deleted, substituted, or added.
Variants may be naturally occurring allelic variants, or non-naturally occurring
variants. Variants may be from the same or from other species and may
encompass homologues, paralogues and orthologues. In certain embodiments,
variants of the polypeptides and polynucleotides disclosed herein possess
biological activities that are the same or similar to those of the disclosed
polypeptides or polypeptides. The term "variant" with reference to polypeptides
and polynucleotides encompasses all forms of polypeptides and polynucleotides
as defined herein.
Polynucleotide variants
Variant polynucleotide sequences preferably exhibit at least 50%, more
preferably at least 51%, more preferably at least 52%, more preferably at least
53%, more preferably at least 54%, more preferably at least 55%, more
preferably at least 56%, more preferably at least 57%, more preferably at least
58%, more preferably at least 59%, more preferably at least 60%, more
preferably at least 61%, more preferably at least 62%, more preferably at least
63%, more preferably at least 64%, more preferably at least 65%, more
preferably at least 66%, more preferably at least 67%, more preferably at least
68%, more preferably at least 69%, more preferably at least 70%, more
preferably at least 71%, more preferably at least 72%, more preferably at least
73%, more preferably at least 74%, more preferably at least 75%, more
preferably at least 76%, more preferably at least 77%, more preferably at least
78%, more preferably at least 79%, more preferably at least 80%, more
preferably at least 81%, more preferably at least 82%, more preferably at least
83%, more preferably at least 84%, more preferably at least 85%, more
preferably at least 86%, more preferably at least 87%, more preferably at least
88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more preferably at least
98%, and most preferably at least 99% identity to a sequence of the present
invention. Identity is found over a comparison window of at least 20 nucleotide
positions, preferably at least 50 nucleotide positions, more preferably at least
100 nucleotide positions, and most preferably over the entire length of a
polynucleotide of the invention.
Polynucleotide sequence identity can be determined in the following manner.
The subject polynucleotide sequence is compared to a candidate polynucleotide
sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov
2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2
sequences - a new tool for comparing protein and nucleotide sequences", FEMS
Microbiol Lett. 174:247-250), which is publicly available from NCBI
(ftp://ftp.ncbi.nih.gov/blast/). In one embodiment the default parameters of
bl2seq are utilized. In a further embodiment the default parameters of bl2seq
are utilized, except that filtering of low complexity parts should be turned off.
Polynucleotide sequence identity may also be calculated over the entire length
of the overlap between a candidate and subject polynucleotide sequences using
global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D.
(1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-
Wunsch global alignment algorithm is found in the needle program in the
EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS: The European
Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16,
No 6. pp. 276-277) which can be obtained from
http://wwwhgmpmrcacuk/Software/EMBOSS/. The
European Bioinformatics Institute server also provides the facility to perform
EMBOSS-needle global alignments between two sequences on line at
http:/wwwebiacuk/em boss/align/.
Alternatively the GAP program may be used which computes an optimal global
alignment of two sequences without penalizing terminal gaps. GAP is described
in the following paper: Huang, X. (1994) On Global Sequence Alignment.
Computer Applications in the Biosciences 10, 227-235.
A preferred method for calculating polynucleotide % sequence identity is based
on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998,
Trends Biochem. Sci. 23, 403-5.)
Polynucleotide variants of the present invention also encompass those which
exhibit a similarity to one or more of the specifically identified sequences that is
likely to preserve the functional equivalence of those sequences and which could
not reasonably be expected to have occurred by random chance. Such sequence
similarity with respect to polypeptides may be determined using the publicly
available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov
2002]) from NCBI (ftp://ftpncbinihgov/blast/).
Alternatively, variant polynucleotides of the present invention hybridize to the
specified polynucleotide sequences, or complements thereof under stringent
conditions.
The term "hybridize under stringent conditions", and grammatical equivalents
thereof, refers to the ability of a polynucleotide molecule to hybridize to a target
polynucleotide molecule (such as a target polynucleotide molecule immobilized
on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined
conditions of temperature and salt concentration. The ability to hybridize under
stringent hybridization conditions can be determined by initially hybridizing
under less stringent conditions then increasing the stringency to the desired
stringency.
With respect to polynucleotide molecules greater than about 100 bases in
length, typical stringent hybridization conditions are no more than 25 to 30o C
(for example, 10o C) below the melting temperature (Tm) of the native duplex
(see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory
Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current
Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide
molecules greater than about 100 bases can be calculated by the formula Tm =
81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular
Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and
McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide
of greater than 100 bases in length would be hybridization conditions such as
prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65oC, 6X SSC,
0.2% SDS overnight; followed by two washes of 30 minutes each in I X SSC,
0.1% SDS at 65o C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS
at 65oC.
With respect to polynucleotide molecules having a length less than 100 bases,
exemplary stringent hybridization conditions are 5 to 10o C below Tm. On
average, the Tm of a polynucleotide molecule of length less than 100 bp is
reduced by approximately (500/oligonucleotide length)o C.
With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen
et al., Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than
those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the
formula described in Giesen et al., Nucleic Acids Res. 1998 Nov 1;26(21):5004-
6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a
length less than 100 bases are 5 to 10o C below the Tm.
Variant polynucleotides of the present invention also encompasses
polynucleotides that differ from the sequences of the invention but that, as a
consequence of the degeneracy of the genetic code, encode a polypeptide
having similar activity to a polypeptide encoded by a polynucleotide of the
present invention. A sequence alteration that does not change the amino acid
sequence of the polypeptide is a "silent variation". Except for ATG (methionine)
and TGG (tryptophan), other codons for the same amino acid may be changed
by art recognized techniques, e.g., to optimize codon expression in a particular
host organism.
Polynucleotide sequence alterations resulting in conservative substitutions of
one or several amino acids in the encoded polypeptide sequence without
significantly altering its biological activity are also included in the invention. A
skilled artisan will be aware of methods for making phenotypically silent amino
acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
Variant polynucleotides due to silent variations and conservative substitutions in
the encoded polypeptide sequence may be determined using the publicly
available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov
2002]) from NCBI (ftp://ftpncbinihgov/blast/) via the tblastx
algorithm as previously described.
Polypeptide variants
The term "variant" with reference to polypeptides encompasses naturally
occurring, recombinantly and synthetically produced polypeptides. Variant
polypeptide sequences preferably exhibit at least 50%, more preferably at least
51%, more preferably at least 52%, more preferably at least 53%, more
preferably at least 54%, more preferably at least 55%, more preferably at least
56%, more preferably at least 57%, more preferably at least 58%, more
preferably at least 59%, more preferably at least 60%, more preferably at least
61%, more preferably at least 62%, more preferably at least 63%, more
preferably at least 64%, more preferably at least 65%, more preferably at least
66%, more preferably at least 67%, more preferably at least 68%, more
preferably at least 69%, more preferably at least 70%, more preferably at least
71%, more preferably at least 72%, more preferably at least 73%, more
preferably at least 74%, more preferably at least 75%, more preferably at least
76%, more preferably at least 77%, more preferably at least 78%, more
preferably at least 79%, more preferably at least 80%, more preferably at least
81%, more preferably at least 82%, more preferably at least 83%, more
preferably at least 84%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more preferably at least
91%, more preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more preferably at least
96%, more preferably at least 97%, more preferably at least 98%, and most
preferably at least 99% identity to a sequences of the present invention.
Identity is found over a comparison window of at least 20 amino acid positions,
preferably at least 50 amino acid positions, more preferably at least 100 amino
acid positions, and most preferably over the entire length of a polypeptide of
the invention.
Polypeptide sequence identity can be determined in the following manner. The
subject polypeptide sequence is compared to a candidate polypeptide sequence
using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in
bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). In
one embodiment the default parameters of bl2seq are utilized. In a further
except the default parameters of bl2seq are utilized, except that filtering of low
complexity parts should be turned off.
Polypeptide sequence identity may also be calculated over the entire length of
the overlap between a candidate and subject polynucleotide sequences using
global sequence alignment programs. EMBOSS-needle (available at
http:/wwwebiacuk/emboss/align/) and GAP (Huang, X.
(1994) On Global Sequence Alignment. Computer Applications in the
Biosciences 10, 227-235.) as discussed above are also suitable global sequence
alignment programs for calculating polypeptide sequence identity.
A preferred method for calculating polypeptide % sequence identity is based on
aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998,
Trends Biochem. Sci. 23, 403-5.)
A variant polypeptide includes a polypeptide wherein the amino acid sequence
differs from a polypeptide herein by one or more conservative amino acid
substitutions, deletions, additions or insertions which do not affect the biological
activity of the peptide. Conservative substitutions typically include the
substitution of one amino acid for another with similar characteristics, e.g.,
substitutions within the following groups: valine, glycine; glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagines, glutamine;
serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
Non-conservative substitutions will entail exchanging a member of one of these
classes for a member of another class.
Analysis of evolved biological sequences has shown that not all sequence
changes are equally likely, reflecting at least in part the differences in
conservative versus non-conservative substitutions at a biological level. For
example, certain amino acid substitutions may occur frequently, whereas others
are very rare. Evolutionary changes or substitutions in amino acid residues can
be modelled by a scoring matrix also referred to as a substitution matrix. Such
matrices are used in bioinformatics analysis to identify relationships between
sequences, one example being the BLOSUM62 matrix shown below (Table 1).
Table 1: The BLOSUM62 matrix containing all possible substitution scores
[Henikoff and Henikoff, 1992].
E L . S
A .4- 4 - 2 - 1 - 4 -2 - i -2 0
Z i 3 1
3 - 3 2 -4
1 4 - 3 4 . -4. .
0 - . 4 3 4 -S - i 4 3 - -2 ϊ -- -2
- 5 - 3 -2 - 3 - ¾ -
3 - 3 -2
· -.5 4 2 : 2 -2 - 3
0 1 ί - 3 4 . ~
- -3 -4 - - .
- 4 -4 4 0 - 3? i.
- · ·Z .-. 3 2 - 3
-2 - -2 2 - 1 - - i
- 4 --3 » Q ·3· 4 -2 X - i
4 ,4 - 1 .
1 4 - - 3
- -X 4 - -2 - 5 2 · 4
¾ -3 -4 ,.J - - -4 - 3 , .
-4 4
- -5 - 2 -2 5 2. ί - · - i
The BLOSUM62 matrix shown is used to generate a score for each aligned
amino acid pair found at the intersection of the corresponding column and row.
For example, the substitution score from a glutamic acid residue (E) to an
aspartic acid residue (D) is 2. The diagonal show scores for amino acids which
have not changed. Most substitutions changes have a negative score. The
matrix contains only whole numbers.
Determination of an appropriate scoring matrix to produce the best alignment
for a given set of sequences is believed to be within the skill of in the art. The
BLOSUM62 matrix in Table 1 is also used as the default matrix in BLAST
searches, although not limited thereto.
Other variants include peptides with modifications which influence peptide
stability. Such analogs may contain, for example, one or more non-peptide
bonds (which replace the peptide bonds) in the peptide sequence. Also included
are analogs that include residues other than naturally occurring L-amino acids,
e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta
or gamma amino acids and cyclic analogs
Constructs, vectors and components thereof
The term "genetic construct" refers to a polynucleotide molecule, usually doublestranded
DNA, which may have inserted into it another polynucleotide molecule
(the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule.
A genetic construct may contain the necessary elements that permit transcribing
the insert polynucleotide molecule, and, optionally, translating the transcript into
a polypeptide. The insert polynucleotide molecule may be derived from the host
cell, or may be derived from a different cell or organism and/or may be a
recombinant polynucleotide. Once inside the host cell the genetic construct may
become integrated in the host chromosomal DNA. The genetic construct may be
linked to a vector.
The term "vector" refers to a polynucleotide molecule, usually double stranded
DNA, which is used to transport the genetic construct into a host cell. The vector
may be capable of replication in at least one additional host system, such as E.
coli.
The term "expression construct" refers to a genetic construct that includes the
necessary elements that permit transcribing the insert polynucleotide molecule,
and, optionally, translating the transcript into a polypeptide. An expression
construct typically comprises in a 5' to 3' direction:
a) a promoter functional in the host cell into which the construct will
be transformed,
b) the polynucleotide to be expressed, and
c) a terminator functional in the host cell into which the construct will
be transformed.
The term "coding region" or "open reading frame" (ORF) refers to the sense
strand of a genomic DNA sequence or a cDNA sequence that is capable of
producing a transcription product and/or a polypeptide under the control of
appropriate regulatory sequences. The coding sequence is identified by the
presence of a 5' translation start codon and a 3' translation stop codon. When
inserted into a genetic construct, a "coding sequence" is capable of being
expressed when it is operably linked to promoter and terminator sequences.
"Operably-linked" means that the sequenced to be expressed is placed under the
control of regulatory elements that include promoters, tissue-specific regulatory
elements, temporal regulatory elements, enhancers, repressors and terminators.
The term "noncoding region" refers to untranslated sequences that are upstream
of the translational start site and downstream of the translational stop site.
These sequences are also referred to respectively as the 5' UTR and the 3' UTR.
These regions include elements required for transcription initiation and
termination and for regulation of translation efficiency.
Terminators are sequences, which terminate transcription, and are found in the 3'
untranslated ends of genes downstream of the translated sequence. Terminators
are important determinants of mRNA stability and in some cases have been found
to have spatial regulatory functions.
The term "promoter" refers to nontranscribed cis-regulatory elements upstream
of the coding region that regulate gene transcription. Promoters comprise cisinitiator
elements which specify the transcription initiation site and conserved
boxes such as the TATA box, and motifs that are bound by transcription factors.
A promoter may be homologous with respect to the polynucleotide to be
expressed. This means that the promoter and polynucleotide are found operably
linked in nature.
Alternatively the promoter may be heterologous with respect to the
polynucleotide to be expressed. This means that the promoter and the
polynucleotide are not found operably linked in nature.
A "transgene" is a polynucleotide that is introduced into an organism by
transformation. The transgene may be derived from the same species or from a
different species as the species of the organism into which the transgene is
introduced. The transgene may also be synthetic and not found in nature in any
species.
A "transgenic plant" refers to a plant which contains new genetic material as a
result of genetic manipulation or transformation. The new genetic material may
be derived from a plant of the same species as the resulting transgenic plant or
from a different species, or may be synthetic.
Preferably the "transgenic" is different from any plant found in nature due the
presence of the transgene.
An "inverted repeat" is a sequence that is repeated, where the second half of the
repeat is in the complementary strand, e.g. :
(5')GATCTA TAGATC(3')
(3')CTAGAT ATCTAG(5')
Read-through transcription will produce a transcript that undergoes
complementary base-pairing to form a hairpin structure provided that there is a
3-5 bp spacer between the repeated regions. The spacer can be any
polynucleotide sequence but is typically at least 3 base pairs in length.
Host cells
Host cells may be derived from, for example, bacterial, fungal, insect, mammalian
or plant organisms.
Methods for isolating or producing polynucleotides
The polynucleotide molecules of the invention can be isolated by using a variety
of techniques known to those of ordinary skill in the art. By way of example,
such polypeptides can be isolated through use of the polymerase chain reaction
(PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction,
Birkhauser, incorporated herein by reference. The polypeptides of the invention
can be amplified using primers, as defined herein, derived from the
polynucleotide sequences of the invention.
Further methods for isolating polynucleotides of the invention include use of all,
or portions of, the polypeptides having the sequence set forth herein as
hybridization probes. The technique of hybridizing labeled polynucleotide probes
to polynucleotides immobilized on solid supports such as nitrocellulose filters or
nylon membranes, can be used to screen the genomic or cDNA libraries.
Exemplary hybridization and wash conditions are: hybridization for 20 hours at
65°C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution;
washing (three washes of twenty minutes each at 55°C) in 1. 0 X SSC, 1% (w/v)
sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5 X
SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C. An optional further wash (for
twenty minutes) can be conducted under conditions of 0. I X SSC, 1% (w/v)
sodium dodecyl sulfate, at 60°C.
The polynucleotide fragments of the invention may be produced by techniques
well-known in the art such as restriction endonuclease digestion, oligonucleotide
synthesis and PCR amplification.
A partial polynucleotide sequence may be used, in methods well-known in the art
to identify the corresponding full length polynucleotide sequence. Such methods
include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218:
340-56) and hybridization- based method, computer/database -based methods.
Further, by way of example, inverse PCR permits acquisition of unknown
sequences, flanking the polynucleotide sequences disclosed herein, starting with
primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16,
8186, incorporated herein by reference). The method uses several restriction
enzymes to generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used as a PCR
template. Divergent primers are designed from the known region. In order to
physically assemble full-length clones, standard molecular biology approaches can
be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Press, 1987).
It may be beneficial, when producing a transgenic plant from a particular species,
to transform such a plant with a sequence or sequences derived from that
species. The benefit may be to alleviate public concerns regarding cross-species
transformation in generating transgenic organisms. Additionally when downregulation
of a gene is the desired result, it may be necessary to utilise a
sequence identical (or at least highly similar) to that in the plant, for which
reduced expression is desired. For these reasons among others, it is desirable to
be able to identify and isolate orthologues of a particular gene in several different
plant species.
Variants (including orthologues) may be identified by the methods described.
Methods for identifying variants
Physical methods
Variant polypeptides may be identified using PCR-based methods (Mullis et al.,
Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the
polynucleotide sequence of a primer, useful to amplify variants of polynucleotide
molecules of the invention by PCR, may be based on a sequence encoding a
conserved region of the corresponding amino acid sequence.
Alternatively library screening methods, well known to those skilled in the art,
may be employed (Sambrook e a/., Molecular Cloning: A Laboratory Manual, 2nd
Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe
sequence, hybridization and/or wash stringency will typically be reduced relatively
to when exact sequence matches are sought.
Polypeptide variants may also be identified by physical methods, for example by
screening expression libraries using antibodies raised against polypeptides of the
invention (Sambrook e a/., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold
Spring Harbor Press, 1987) or by identifying polypeptides from natural sources
with the aid of such antibodies.
Computer based methods
The variant sequences of the invention, including both polynucleotide and
polypeptide variants, may also be identified by computer-based methods wellknown
to those skilled in the art, using public domain sequence alignment
algorithms and sequence similarity search tools to search sequence databases
(public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others).
See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online
resources. Similarity searches retrieve and align target sequences for comparison
with a sequence to be analyzed (i.e., a query sequence). Sequence comparison
algorithms use scoring matrices to assign an overall score to each of the
alignments.
An exemplary family of programs useful for identifying variants in sequence
databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including
BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available
from (ftp://ftpncbinihgov/blast/) or from the National Center
for Biotechnology Information (NCBI), National Library of Medicine, Building 38A,
Room 8N805, Bethesda, MD 20894 USA. The NCBI server also provides the
facility to use the programs to screen a number of publicly available sequence
databases. BLASTN compares a nucleotide query sequence against a nucleotide
sequence database. BLASTP compares an amino acid query sequence against a
protein sequence database. BLASTX compares a nucleotide query sequence
translated in all reading frames against a protein sequence database. tBLASTN
compares a protein query sequence against a nucleotide sequence database
dynamically translated in all reading frames. tBLASTX compares the six-frame
translations of a nucleotide query sequence against the six-frame translations of a
nucleotide sequence database. The BLAST programs may be used with default
parameters or the parameters may be altered as required to refine the screen.
The use of the BLAST family of algorithms, including BLASTN, BLASTP, and
BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25:
3389-3402, 1997.
The "hits" to one or more database sequences by a queried sequence produced
by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align
and identify similar portions of sequences. The hits are arranged in order of the
degree of similarity and the length of sequence overlap. Hits to a database
sequence generally represent an overlap over only a fraction of the sequence
length of the queried sequence.
The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce
"Expect" values for alignments. The Expect value (E) indicates the number of hits
one can "expect" to see by chance when searching a database of the same size
containing random contiguous sequences. The Expect value is used as a
significance threshold for determining whether the hit to a database indicates true
similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is
interpreted as meaning that in a database of the size of the database screened,
one might expect to see 0.1 matches over the aligned portion of the sequence
with a similar score simply by chance. For sequences having an E value of 0.01
or less over aligned and matched portions, the probability of finding a match by
chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX,
tBLASTN or tBLASTX algorithm.
Multiple sequence alignments of a group of related sequences can be carried out
with CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, TJ. (1994)
CLUSTALW: improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, positions-specific gap penalties and weight matrix
choice. Nucleic Acids Research, 22:4673-4680, http://www-igbmcustrasbg<
dot>fr/BioInfo/ClustalW/Top.html) or T-COFFEE (Cedric Notredame,
Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and
accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or
PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987,
J. Mol. Evol. 25, 351).
Pattern recognition software applications are available for finding motifs or
signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds
motifs and signature sequences in a set of sequences, and MAST (Motif Alignment
and Search Tool) uses these motifs to identify similar or the same motifs in query
sequences. The MAST results are provided as a series of alignments with
appropriate statistical data and a visual overview of the motifs found. MEME and
MAST were developed at the University of California, San Diego.
PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et
a/., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of
uncharacterized proteins translated from genomic or cDNA sequences. The
PROSITE database (wwwexpasyorg/prosite) contains biologically
significant patterns and profiles and is designed so that it can be used with
appropriate computational tools to assign a new sequence to a known family of
proteins or to determine which known domain(s) are present in the sequence
(Falquet et a/., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can
search SWISS-PROT and EMBL databases with a given sequence pattern or
signature.
Methods for isolating polypeptides
The polypeptides of the invention, or used in the methods of the invention,
including variant polypeptides, may be prepared using peptide synthesis methods
well known in the art such as direct peptide synthesis using solid phase
techniques (e.g. Stewart et a/., 1969, in Solid-Phase Peptide Synthesis, WH
Freeman Co, San Francisco California, or automated synthesis, for example using
an Applied Biosystems 431A Peptide Synthesizer (Foster City, California).
Mutated forms of the polypeptides may also be produced during such syntheses.
The polypeptides and variant polypeptides of the invention, or used in the
methods of the invention, may also be purified from natural sources using a
variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed,
Methods in Enzymology, Vol. 182, Guide to Protein Purification,).
Alternatively the polypeptides and variant polypeptides of the invention, or used
in the methods of the invention, may be expressed recombinantly in suitable host
cells and separated from the cells as discussed below.
Methods for producing constructs and vectors
The genetic constructs of the present invention comprise one or more
polynucleotide sequences of the invention and/or polynucleotides encoding
polypeptides of the invention, and may be useful for transforming, for example,
bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs
of the invention are intended to include expression constructs as herein defined.
Methods for producing and using genetic constructs and vectors are well known in
the art and are described generally in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for producing host cells comprising polynucleotides, constructs or vectors
The invention provides a host cell which comprises a genetic construct or vector
of the invention.
Host cells comprising genetic constructs, such as expression constructs, of the
invention are useful in methods well known in the art (e.g. Sambrook et al.,
Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987
; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987)
for recombinant production of polypeptides of the invention. Such methods may
involve the culture of host cells in an appropriate medium in conditions suitable
for or conducive to expression of a polypeptide of the invention. The expressed
recombinant polypeptide, which may optionally be secreted into the culture, may
then be separated from the medium, host cells or culture medium by methods
well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182,
Guide to Protein Purification).
Methods for producing plant cells and plants comprising constructs and vectors
The invention further provides plant cells which comprise a genetic construct of
the invention, and plant cells modified to alter expression of a polynucleotide or
polypeptide of the invention, or used in the methods of the invention. Plants
comprising such cells also form an aspect of the invention.
Methods for transforming plant cells, plants and portions thereof with
polypeptides are described in Draper et al., 1988, Plant Genetic Transformation
and Gene Expression. A Laboratory ManuaL Blackwell Sci. Pub. Oxford, p. 365;
Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag,
Berlin.; and Gelvin e a/., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub.
Dordrecht. A review of transgenic plants, including transformation techniques, is
provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press,
London.
Methods for genetic manipulation of plants
A number of plant transformation strategies are available (e.g. Birch, 1997, Ann
Rev Plant Phys Plant Mol Biol, 48, 297, Hellens RP, et al (2000) Plant Mol Biol 42:
819-32, Hellens R et al Plant Meth 1: 13). For example, strategies may be
designed to increase expression of a polynucleotide/polypeptide in a plant cell,
organ and/or at a particular developmental stage where/when it is normally
expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue,
organ and/or at a particular developmental stage which/when it is not normally
expressed. The expressed polynucleotide/polypeptide may be derived from the
plant species to be transformed or may be derived from a different plant species.
Genetic constructs for expression of genes in transgenic plants typically include
promoters for driving the expression of one or more cloned polynucleotide,
terminators and selectable marker sequences to detect presence of the genetic
construct in the transformed plant.
The promoters suitable for use in genetic constructs may be functional in a cell,
tissue or organ of a monocot or dicot plant and include cell-, tissue- and organspecific
promoters, cell cycle specific promoters, temporal promoters, inducible
promoters, constitutive promoters that are active in most plant tissues, and
recombinant promoters. Choice of promoter will depend upon the temporal and
spatial expression of the cloned polynucleotide, so desired. The promoters may
be those normally associated with a transgene of interest, or promoters which are
derived from genes of other plants, viruses, and plant pathogenic bacteria and
fungi. Those skilled in the art will, without undue experimentation, be able to
select promoters that are suitable for use in modifying and modulating plant traits
using genetic constructs comprising the polynucleotide sequences of the
invention. Examples of constitutive plant promoters include the CaMV 35S
promoter, the nopaline synthase promoter and the octopine synthase promoter,
and the Ubi 1 promoter from maize. Plant promoters which are active in specific
tissues, respond to internal developmental signals or external abiotic or biotic
stresses are described in the scientific literature. Exemplary promoters are
described, e.g., in WO 02/00894, which is herein incorporated by reference.
Exemplary terminators that are commonly used in plant transformation genetic
construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the
Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators,
the Zea mays zein gene terminator, the Oryza sativa ADP-glucose
pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
Selectable markers commonly used in plant transformation include the neomycin
phophotransferase I I gene (NPT II) which confers kanamycin resistance, the
aadA gene, which confers spectinomycin and streptomycin resistance, the
phosphinothricin acetyl transferase {bar gene) for Ignite (AgrEvo) and Basta
(Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for
hygromycin resistance.
Use of genetic constructs comprising reporter genes (coding sequences which
express an activity that is foreign to the host, usually an enzymatic activity
and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for
promoter expression analysis in plants and plant tissues are also contemplated.
The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature
303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T.,
Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
Gene silencing
Transformation strategies may be designed to reduce expression of a
polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular
developmental stage which/when it is normally expressed. Such strategies are
known as gene silencing strategies.
Gene silencing strategies may be focused on the gene itself or regulatory
elements which effect expression of the encoded polypeptide. "Regulatory
elements" is used here in the widest possible sense and includes other genes
which interact with the gene of interest.
Genetic constructs designed to decrease or silence the expression of a
polynucleotide/polypeptide of the invention may include an antisense copy of a
polynucleotide of the invention. In such constructs the polynucleotide is placed in
an antisense orientation with respect to the promoter and terminator.
An "antisense" polynucleotide is obtained by inverting a polynucleotide or a
segment of the polynucleotide so that the transcript produced will be
complementary to the mRNA transcript of the gene, e.g.,
5'GATCTA 3' (coding strand) 3'CTAGAT 5' (antisense strand)
3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA
Genetic constructs designed for gene silencing may also include an inverted
repeat. An 'inverted repeat' is a sequence that is repeated where the second half
of the repeat is in the complementary strand, e.g.,
5'-GATCTA TAGATC-3'
3'-CTAGAT ATCTAG-5'
The transcript formed may undergo complementary base pairing to form a hairpin
structure. Usually a spacer of at least 3-5 bp between the repeated region is
required to allow hairpin formation. Constructs including such invented repeat
sequences may be used in RNA interference (RNAi) and therefore can be referred
to as RNAi constructs.
Another silencing approach involves the use of a small antisense RNA targeted to
the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053).
Use of such small antisense RNA corresponding to polynucleotide of the invention
is expressly contemplated.
The term genetic construct as used herein also includes small antisense RNAs and
other such polypeptides effecting gene silencing.
Transformation with an expression construct, as herein defined, may also result in
gene silencing through a process known as sense suppression (e.g. Napoli e a/.,
1990, Plant Cell 2, 279; de Carvalho Niebel e a/., 1995, Plant Cell, 7, 347). In
some cases sense suppression may involve over-expression of the whole or a
partial coding sequence but may also involve expression of non-coding region of
the gene, such as an intron or a 5' or 3' untranslated region (UTR). Chimeric
partial sense constructs can be used to coordinately silence multiple genes
(Abbott et a/., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta
204: 499-505). The use of such sense suppression strategies to silence the
expression of a polynucleotide of the invention is also contemplated.
The polynucleotide inserts in genetic constructs designed for gene silencing may
correspond to coding sequence and/or non-coding sequence, such as promoter
and/or intron and/or 5' or 3' UTR sequence, of the corresponding gene.
Other gene silencing strategies include dominant negative approaches and the
use of ribozyme constructs (Mclntyre, 1996, Transgenic Res, 5, 257).
Pre-transcriptional silencing may be brought about through mutation of the gene
itself or its regulatory elements. Such mutations may include point mutations,
frameshifts, insertions, deletions and substitutions.
Transformation protocols
The following are representative publications disclosing genetic transformation
protocols that can be used to genetically transform the following plant species:
Rice (Alam e a/., 1999, Plant Cell Rep. 18, 572); apple (Yao e a/., 1995, Plant
Cell Reports 14, 407-412); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981,
840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (US
Patent Serial No. 5, 159, 135); potato (Kumar et al., 1996 Plant J. 9, : 821);
cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al.,
1987, Plant Cell Rep. 6, 439); tobacco (Horsch e a/., 1985, Science 227, 1229);
cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent
Nos. 5, 187, 073 and 6. 020, 539); peppermint (Niu e a/., 1998, Plant Cell Rep.
17, 165); citrus plants (Pena e a/., 1995, Plant Sci.104, 183); caraway (Krens et
al., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935);
soybean (US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455
and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US
Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6,
037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871);
cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant Cell Rep.
24(1):45-51); Prunus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song
and Sink 2005 Plant Cell Rep. 2006 ;25(2): 117-23; Gonzalez Padilla et al., 2003
Plant Cell Rep.22(l):38-45); strawberry (Oosumi et al., 2006 Planta.
223(6): 1219-30; Folta et al., 2006 Planta Apr 14; PMID: 16614818), rose (Li et
al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995;44: 129-33),
tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al.,
1995, Plant Cell Rep. 14, 407-412) and Actinidia eriantha (Wang et al., 2006,
Plant Cell Rep. 25,5: 425-31), silver birch (Keinonen-Mettala et al., 1998, Plant
Cell Rep. 17: 356-361.) and aspen (Nilsson O, et al., 1992, Transgenic Research.
1: 209-220). Transformation of other species is also contemplated by the
invention. Suitable methods and protocols are available in the scientific
literature.
Several further methods known in the art may be employed to alter expression of
activity of a nucleotide and/or polypeptide of the invention. Such methods
include but are not limited to Tilling (Till e a/., 2003, Methods Mol Biol, 2%, 205),
so called "Deletagene" technology (Li et al., 2001, Plant Journal 27(3), 235) and
the use of artificial transcription factors such as synthetic zinc finger transcription
factors, (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally
antibodies or fragments thereof, targeted to a particular polypeptide may also be
expressed in plants to modulate the activity of that polypeptide (Jobling et al.,
2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be
applied. Additionally peptides interacting with a polypeptide of the invention may
be identified through technologies such as phase-display (Dyax Corporation).
Such interacting peptides may be expressed in or applied to a plant to affect
activity of a polypeptide of the invention. Use of each of the above approaches in
alteration of expression of a nucleotide and/or polypeptide of the invention is
specifically contemplated.
The terms "to alter expression of" and "altered expression" of a polynucleotide or
polypeptide of the invention, or used in the methods of the invention, are
intended to encompass the situation where genomic DNA corresponding to a
polynucleotide of the invention is modified thus leading to altered expression of a
polynucleotide or polypeptide of the invention. Modification of the genomic DNA
may be through genetic transformation or other methods known in the art for
inducing mutations. The "altered expression" can be related to an increase or
decrease in the amount of messenger RNA and/or polypeptide produced and may
also result in altered activity of a polypeptide due to alterations in the sequence
of a polynucleotide and polypeptide produced.
Methods of selecting plants
Methods are also provided for selecting plants with increased leaf or root
biomass. Such methods involve testing of plants for altered for the expression of
at least one PEAPOD polynucleotide or polypeptide, including those as defined or
disclosed herein. Such methods may be applied at a young age or early
developmental stage when the increased leaf or root biomass characteristics may
not necessarily be easily measurable.
The expression of a polynucleotide, such as a messenger RNA, is often used as an
indicator of expression of a corresponding polypeptide. Exemplary methods for
measuring the expression of a polynucleotide include but are not limited to
Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
Polynucleotides or portions of the polynucleotides of the invention are thus useful
as probes or primers, as herein defined, in methods for the identification of plants
with increased leaf or root biomass. The polynucleotides of the invention, or
disclosed herein, may be used as probes in hybridization experiments, or as
primers in PCR based experiments, designed to identify such plants.
Alternatively antibodies may be raised against PEAPOD polypeptides as described
or disclosed herein Methods for raising and using antibodies are standard in the
art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold
Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to
detect altered expression of such polypeptides. Such methods may include ELISA
(Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western
analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
These approaches for analysis of polynucleotide or polypeptide expression and
the selection of plants with increased leaf or root biomass are useful in
conventional breeding programs designed to produce varieties with such altered
characteristics.
Plants
The term "plant" is intended to include a whole plant, any part of a plant,
propagules and progeny of a plant.
The term 'propagule' means any part of a plant that may be used in reproduction
or propagation, either sexual or asexual, including seeds and cuttings.
The plants of the invention may be grown and either self-ed or crossed with a
different plant strain and the resulting hybrids, with the desired phenotypic
characteristics, may be identified. Two or more generations may be grown to
ensure that the subject phenotypic characteristics are stably maintained and
inherited. Plants resulting from such standard breeding approaches also form an
aspect of the present invention.
Control of plant growth and development by Gibberellins (GA), Brassinosteroids
(BR) and other plant hormones
Gibberellins (GA) and Brassinosteroids (BR) are two classes of plant hormones;
between them they are involved in many aspects of plant morphogenesis and
growth; including: seed germination, cell elongation, vascular development, see
size, leaf erectness, flowering, leaf and fruit senescence (Mathew et al 2009,
NZJAR 52, 213-225; Hou et al 2010, Developmental Cell 19, 884-894; Jiang and
Lin 2013, Plant Signaling and Behaviour 8: 10, e25928).
Given their roles in plant development the ability to manipulate either the levels
of GA and BR or their downstream targets is highly desirable in terms of
improving both yield and quality in many plant species. Indeed there are some
commercial examples where exogenous applications of either hormone are used
to improve agronomic value.
GA can be applied to ryegrass pasture to stimulate out-of-season growth as well
as promote flowering (Mathew et al 2009, NZJAR 52, 213-225), it can also be
used to counteract the adverse effects of cooler temperatures on sugarcane (a
tropical C4 grass). GAs are also used to enlarge fruit size of seedless grapes and
cherries, to promote fruit set in apple and pear and to delay rind-aging in
particular citrus crops (Sun 2011, Current Biology 21, R338-R345). Similarly, BR
preparations are recommended for improving crop yield and quality of tomato,
potato, cucumber, pepper and barley, rice, maize, wheat, cotton, and tobacco
(Prusakova et al 1999, Agrarian Russia, 41-44; Khripach et al 2000 Annals of
Botany 86, 441-447; Anjum et al 2011 J. Agronomy Crop Sci. 197, 177-185;
Vardhini 2012 J. Phytology 4, 1-3). However, the low adoption of commercially
applied brassinosteroids may reflect the cost and the fact that plants do not
efficiently absorb steroids when they are applied exogenously. In addition, the
need to strictly control timing and concentration of exogenous supplied GA and
BR limits their applications.
For the most part the GA and BR biosynthesis and catabolic pathways in
angiosperms have been characterized and include negative regulators and
downstream transcription factor targets. Upon binding GA or BR to their
respective receptor a complex signal pathway ensues and in both cases a central
point of regulation involves the ubiquitin-proteasome pathway altering the level
of the negative regulator DELLA (in the case of GA) and the transcriptional
regulator BZR1 (in the case of BR).
The removal of DELLA proteins results in the removal of growth repression and
promotion of GA-responsive growth and development. Conversely the detection
of BR leads to the accumulation of unphosphorylated BZR1 protein in the nucleus.
Dephosphorylation of BZR1 prevents its degradation by the proteasome and
instead allows the binding of BZR1 with other DNA binding transcription factors
and interacts with transcriptional cofactors. This leads to the regulation of
thousands of genes involved in growth and other cellular processes, including the
inhibition of expression of BR biosynthetic genes (He et al 2005, Science 307,
1634-1638; Guo et al 2013, Current Opinion Plant Biol. 16, 545-553).
There are a number of endogenous signals and environmental cues that influence
the GA-GID1-DELLA regulatory module in which DELLA integrates different
signalling activities by direct protein-protein interaction with multiple key
regulatory proteins from other pathways. As such DELLA proteins are master
growth repressors that control plant growth and development by integrating
internal signals from other hormone pathways (auxin, abscisic acid, jasmonic acid
and ethylene), and external biotic (pathogen) and abiotic (light conditions, cold
and salt stresses) cues (Sun 2011, Current Biology 21, R338-R345). Drought is
one of the most important environmental constraints limiting plant growth and
agricultural productivity. Unsurprisingly, there is a positive correlation between
improved drought tolerance with a more extensive root system including deeper
roots and more lateral roots both of which enable soil exploration and belowground
resources acquisition (Yu et al 2008, Plant Cell 20, 1134-1151; Werner et
al 2010, Plant Cell 22, 3905-3920). Thus it follows that a common agricultural
target is the optimization of root system architecture in order to help overcome
yield limitations in crop plants caused by water or nutrient shortages. However,
of all the abiotic stresses that curtail crop productivity, drought is the most
devastating one and the most recalcitrant to breeder's efforts. Classic breeding
approaches are difficult because the trait is governed by many genes and is
difficult to score (Werner et al 2010, Plant Cell 22, 3905-3920). While markerassisted
selection (MAS), quantitative trait loci (QTL) and other genomic
approaches are being widely used to assist breeding efforts to produce droughtresilient
cultivars (Tuberosa and Salvi, 2006, Trends in Plant Science, 11 :405-
412) the system is limited to the variation present in the screening population.
Interestingly, rice has only one DELLA protein (SLR1), Maize has two (d8 and
d9)(Lawit et al 2010, Plant Cell Physiol 51, 1854-1868) while Arabidopsis has
five (GA1, RGA, RGL1, RGL2 and RGL3) (Achard and Genschik 2009, J. Exp. Bot.
60, 1085-1092). Furthermore, in a recent phylogenetic analysis it Chen et al
2013 found five out of the six grass species they analysed had only a single
DELLA while 14 out of the 18 dicot species had two or more DELLA proteins. In
contrast, there are 6 members of the BZR family in rice, 10 in maize
(wwwGrassiusorg) and 6 in Arabidopsis (Wang et al 2002,
Developmental Cell 2, 505-513).
The growth and development of plants relies on numerous connections between
signalling pathways that provides the high developmental plasticity demanded by
their sessile life habit (Gallego-Bartolome et al 2012, PNAS 109, 13446-13451).
Thus rather than each hormone-signalling pathway existing as an insulated
module current evidence indicates that there is a high degree of interaction
between different pathways and that a given hormone frequently modulates the
output triggered by the rest. By example, it has recently been shown that the
cross talk between the GA and BR signalling pathways involves direct interaction
between DELLAs and BZR1/BES1 whereby DELLA proteins not only affect the
protein stability but also inhibit the transcriptional activity of BZR1 (Li and He
2013, Plant Signaling and Behaviour 8:7, e24686 and references therein). Thus
the promotion of cell elongation by GA is partly through the removal of the
DELLA-mediated inhibition of BZR1.
It has recently been demonstrated that plant growth and development can be
modified through direct manipulation of the master growth regulators DELLA
(Lawit, Kundu, Rao and Tomes, 2007, Isolated polynucleotide molecules
corresponding to mutant and wild-type alleles of the maize D9 gene and methods
of use, WO 2007124312 A2) and BZR1 (Chory and Wang, 2005, Genes involved
in brassinosteroid hormone action on plants, US 6,921,848 B2).
Steroid hormones play an essential role in the coordination of a wide range of
developmental and physiological processes in both plants and animals (Thummel
and Chory 2002, Genes Dev. 16, 3113-3129). In plants the steroid hormone
brassinosteroid (BR) has extensive effects on growth, development and responses
to both biotic and abiotic stresses (Zhu et al 2013, Development 140, 1615-1620;
Clouse 2011, Plant Cell 23, 1219-1230). In contrast to animal steroid hormone
signalling, which functions through nuclear receptors, in plants BRs bind to the
extracellular domain of the cell surface receptor kinase BRASSINOSTEROID
INSENSITIVE 1 (BRI1) and activate an intracellular signal transduction cascade
that regulates gene expression (Clouse 2011, Plant Cell 23, 1219-1230; Kinoshita
et al 2005, Nature 433, 167-171). There are multiple steps involving activation
and inactivation of intermediates leading to the phosphorylation of two
transcription factors, Brassinazole Resistant 1 (BZR1) and BZR2 (also known as
BES1). Thus the signal transduction BZR transcription factors are the target
components converting signalling into BR responsive gene expression.
There is an emerging pattern in plant hormone signalling where the target
transcription factors activated by hormones are also negatively regulated by
specific repressor complexes. For example, in the jasmonic acid (JA), auxin,
abscisic acid (ABA) and strigolactone (SL) signalling pathways the target
transcription factors are negatively regulated by repressor complexes utilising
TOPLESS (TPL) as a common co-repressor recruited by a hormone pathway
specific repressor (Pauwels et al 2010, Nature 464, 788-791). In the JA
transduction pathway the JASMONATE ZIM DOMAIN (JAZ) family of
transcriptional repressors both interact with the target JA-responsive
transcriptional activator MYC2 and recruit TPL, either directly or via the adaptor
protein Novel Interactor of JAZ (NINJA) (Pauwels et al 2010, Nature 464, 788-
791).
Accordingly, the ability to regulate the GA and BR pathways to influence many
different agricultural traits of interest is of considerable value to commercial
agriculture.
The applicant's invention
As discussed above, the present invention relates to a method for increasing at
least one of leaves and root biomass in Poaceae plants by ectopic expression of
PEAPOD.
Without wishing to be bound by theory, the applicants have shown that PEAPOD
(PPD) appears to be involved in the modulation of both the GA and BR pathways
either through direct or indirect interaction with the master growth regulators
DELLA and BZR.
Analysis of the primary amino acid structure of PPD proteins indicates the
presence of a highly conserved novel plant specific domain present only these
proteins. There are homologues of PPD in a wide range of eudicot, conifers and
some monocot plants (palms, banana, orchids, duckweed) but not Poaceae
(grasses).
The PPD genes of Arabidopsis encode proteins that are members of the plantspecific
TIFY family, named after the core TIF[F/Y]XG motif found within a domain
known as ZIM (Vanholme et al 2007, Trends Plant Sci. 12, 239-244). The two
Arabidopsis PPD proteins, PPDl and PPD2, are included in the same class I I TIFY
group as twelve well characterised JAZ proteins that act as repressors of
jasmonate responses. However, the PPD proteins and the one other non-JAZ
protein in the group do not appear to be involved in responses to jasmonate
hormone signalling (Pauwels et al 2010, Nature 464, 788-791).
Again, without wishing to be bound by theory, the applicants propose that the
increases in leaf and root biomass, according to the invnetion, are mediated by a
new mechanism for regulating both the GA and BR pathways in the Poaceae
family using the PPD gene. Examples 3 and 4 below support this proposal,
This invention may also be said broadly to consist in the parts, elements and
features referred to or indicated in the specification of the application, individually
or collectively, and any or all combinations of any two or more said parts,
elements or features, and where specific integers are mentioned herein which
have known equivalents in the art to which this invention relates, such known
equivalents are deemed to be incorporated herein as if individually set forth.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood with reference to the
accompanying drawings in which are described as follows:
Figure 1A shows the synteny map of flanking genes around the PPD loci in various
dicotyledonous and monocotyledonous plants and the absence of PPD genes in
the same location in the Poaceae.
Figure I B shows the presence of numerous repeats in the rice chromosome where
synteny predicts PPD should have been.
Figure 2 shows the 46 amino acid residues comprising the PEAPOD region from a
range of plant species, identical residues are shown by an asterisk.
Figure 3 shows the internal 27 amino acid residues within the PEAPOD region
from a range of plant species, identical residues are shown by an asterisk.
Figure 4 shows the 6 amino acid residues of the TIFY domain on PEAPOD proteins
from a range of plant species, identical residues are shown by an asterisk.
Figure 5 shows a schematic representation of the PPD protein and the
approximate location of conserved PPD, TIFY and Jas* regions
Figure 6 shows the dimerization of PPD and the interaction between TPL and
NINJA in Y2H assays.
Figure 7 shows the interaction between PPD and NINJA and the interaction
between TPL and BZR1 in Y2H assays.
Figure 8 shows the interaction between PPD, NINJA, TPL and BZR1 in young (A
and B) and old (C) leaves using BiFC assays.
Figure 9 shows a schematic representation of the PPD-NINJA-TPL-BZR1 complex.
Figure 10 shows the interaction between PPD and BZR1 in Y2H assays.
Figure 11 shows the response of Wild Type, Appd mutant, and PEAPOD
overexpressor (PPD-OX) hypocotyl length to exogenous GA and PAC applications.
Figures 12A and 12B show the increase in shoot and root growth of ryegrass
plants over expressing PEAPOD from Arabidopsis thaliana or PEAPOD from
Ambroella trichopoda compared to the wild type and vector control.
Figure 13 shows that the PEAPOD proteins from Arabidopsis thaliana; Picea
sitchensis, Amborella trichopoda, Musa acuminate, Trifolium repens and
Selaginella moellendorffii are functionally equivalent. An optimized PEAPOD
coding sequence from each was used to complement the PEAPOD deletion mutant
Appd Arabidopsis thaliana (ecotype Landsberg erecta). Seedling images were
taken at an equivalent developmental stage.
EXAMPLES
The invention will now be illustrated with reference to the following non-limiting
examples.
Example 1 : Characterisation of PEAPOD genes multiple plant species
To identify PPD gene orthologues in other plant species the conserved PPD region
(46 amino acids) from the Arabidopsis PPD1 gene (SEQ ID NO: 27) was used for
searches of public plant gene sequence databases using the search programmes
TBLASTN and BLASTP (Altschul et al 1990). PEAPOD sequences were identified
from a diverse range of plant species including the mosses, conifers, all orders of
dicotyledonous examined and some of the monocotyledonous orders, including:
palms, bananas, orchids and duckweed. The same search method indicated that
PEAPOD sequences are not found in the grasses. Extensive syntany comparisons
showed that in the poace genomes analysed (Brachypodium distachyon, Oryza
sativa and Zea mays) the region expected to contain PPD genes has been
disrupted (Figure 1A) and now contains numerous repeats (Figure IB).
Representative PEAPOD protein sequences are shown in SEQ ID NO: 1-26 and
nucleic acid sequences are shown in SEQ ID NO:80-104 respectively.
The 46 amino acid PEAPOD region from Arabidopsis thaliana PPDl is shown in
SEQ ID NO: 27. This region from polypeptides SEQ ID NO: 1-was aligned by
vector NTI (VNTI) as shown in FIGURE 2.
SEQ ID NO:28 shows the consensus for this 46 amino acid PPD region. SEQ ID
NO: 29 shows the same consensus region but shows which amino acids can be
present at each of the variable positions.
A 27 amino acid subsequence from within the 46 amino acid PEAPOD region from
Arabidopsis thaliana PPDl is shown in SEQ ID NO:30.
Alignment of this 27 amino acid subsequence for reach of the same sequences as
in Figure 2, is shown in Figure 3.
SEQ ID NO:31 shows the consensus for this 27 amino acid PPD region. SEQ ID
NO: 32 shows the same consensus region but shows which amino acids can be
present at each of the variable positions.
In each of the PPD peptide sequences of SEQ ID NO: 1-26 there is also a
conserved TIFY motif which is located after the 46 amino acid PPD region. The
number of amino acid residues separating the C-terminus of the PPD region and
the N-terminus of the TIFY motif depends on the source of the PPD; for example
the number varies between 46 to 140 amino acids for SEQ ID NO: 1-26.
SEQ ID NO: 33 shows the Arabidopsis PPDl sequence over the TIFY motif. The
alignment of the TIFY motif (as described by Vanholme et al 2007, Trends Plant
Sci. 12, 239-244) from SEQ ID NO: 1-26 is shown in Figure 4.
SEQ ID NO: 34 shows the consensus for this 6 amino acid TIFY motif. SEQ ID
NO: 35 shows the same consensus region but shows which amino acids can be
present at each of the variable positions.
Completely conserved residues in the PPD and TIFY domains are highlighted with
asterisks in Figures 2-4.
The applicants assert that these regions and motifs described above are found in
all PEAPOD proteins identified and are diagnostic for such PEAPOD proteins
Example 2 : Demonstrating PEAPOD functionality of PEAPOD sequences
from multiple plant species
The functionality of any PEAPOD sequence can be confirmed by complementation
of the Arabidopsis Appd mutant leaf phenotype. Complementation of the
Arabidopsis Appd mutant leaf phenotype was first used to identify the Arabidopsis
PPD gene (White 2006). This was seen by a restoration of the wild type flattened
leaf phenotype and normal rosette shape as opposed to the domed leaf and the
twisting of the rosette to a "propeller" phenotype.
PEAPOD sequences, such as those of SEQ IN NO: 1-26 (including: palm, conifer,
moss, orchid and other dicot species) or any other PEAPOD sequence to be tested
can be transformed into the Arabidopsis Appd mutant by methods well known to
those skilled in the art. An example of such a method is described below.
Cloning and Gene Constructs
Generation of CaMV35s: : Arabidopsis thaliana PPDl construct for over expression
of Arabidopsis PPDl in the Arabidopsis Appd mutant
An expression construct was synthesised to enable the over expression of
Arabidopsis thaliana PPDl under the CaMV35s promoter (SEQ ID NO. 129) in the
Arabidopsis Appd mutant. The PPD ORF was optimised for expression in
Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid
Research 15, 6643-6653), optimisation of condons, removal of mRNA instability
sequences, removal of polyA signal sequences, removal of cryptic splice sites,
addition of a BamHl removable C-terminal V5 epitope and His tag tail (encoding
SEQ ID NO: 37) and addition of a double stop codon. The construct (with and
without the tail) was then placed between the CaMV35s promoter and ocs
terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO: 105 and
SEQ ID NO: 111 respectively.
Generation of CaMV35s:: Trifolium repens PPD construct for over expression of
Trifolium repens PPD1 in the Arabidopsis Appd mutant
An expression construct was synthesised to enable the over expression of
Trifolium repens PPD under the CaMV35s promoter (SEQ ID NO. 129) in the
Arabidopsis Appd mutant. The PPD ORF was optimised for expression in
Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid
Research 15, 6643-6653), optimisation of condons, removal of mRNA instability
sequences, removal of polyA signal sequences, removal of cryptic splice sites,
addition of a BamHl removable C-terminal V5 epitope and His tag tail (encoding
SEQ ID NO: 37) and addition of a double stop codon. The construct (with and
without the tail) was then placed between the CaMV35s promoter and ocs
terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO: 106 and
SEQ ID NO: 112 respectively.
Generation of CaMV35s:: Amborella trichopoda PPD construct for over expression
of Amborella trichopoda PPD in the Arabidopsis Appd mutant
An expression construct was synthesised to enable the over expression of
Amborella trichopoda PPD under the CaMV35s promoter (SEQ ID NO. 129) in the
Arabidopsis Appd mutant. The PPD ORF was optimised for expression in
Arabidopsis; this included a modified Joshi sequence (Joshi 1997) , Nucleic Acid
Research 15, 6643-6653, optimisation of condons, removal of mRNA instability
sequences, removal of polyA signal sequences, removal of cryptic splice sites,
addition of a BamHl removable C-terminal V5 epitope and His tag tail (encoding
SEQ ID NO: 37) and addition of a double stop codon. The construct (with and
without the tail) was then placed between the CaMV35s promoter and ocs
terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO: 107 and
SEQ ID NO: 113 respectively.
Generation of CaMV35s:: Musa acuminate PPD construct for over expression of
Musa acuminate PPD in the Arabidopsis Appd mutant
An expression construct was synthesised to enable the over expression of Musa
acuminate PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis
Appd mutant. The PPD ORF was optimised for expression in Arabidopsis; this
included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-
6653), optimisation of condons, removal of mRNA instability sequences, removal
of polyA signal sequences, removal of cryptic splice sites, addition of a BamHl
removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and
addition of a double stop codon. The construct (with and without the tail) was
then placed between the CaMV35s promoter and ocs terminator by the
GATEWAY® LR reaction, which coded for SEQ ID NO: 108 and SEQ ID NO: 114
respectively.
Generation of CaMV35s:: Picea sitchensis PPD1 construct for over expression of
Picea sitchensis PPD in the Arabidopsis Appd mutant
An expression construct was synthesised to enable the over expression of Picea
sitchensis PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis
Appd mutant. The PPD ORF was optimised for expression in Arabidopsis; this
included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-
6653), optimisation of condons, removal of mRNA instability sequences, removal
of polyA signal sequences, removal of cryptic splice sites, addition of a BamHl
removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and
addition of a double stop codon. The construct (with and without the tail) was
then placed between the CaMV35s promoter and ocs terminator by the
GATEWAY® LR reaction, which coded for SEQ ID NO: 109 and SEQ ID NO: 115
respectively.
Generation of CaMV35s:. Selaginella moellendorffii PPD1 construct for over
expression of Selaginella moellendorffii PPD in the Arabidopsis Appd mutant
An expression construct was synthesised to enable the over expression of
Selaginella moellendorffii PPD under the CaMV35s promoter (SEQ ID NO. 129) in
the Arabidopsis Appd mutant. The PPD ORF was optimised for expression in
Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid
Research 15, 6643-6653), optimisation of condons, removal of mRNA instability
sequences, removal of polyA signal sequences, removal of cryptic splice sites,
addition of a BamHl removable C-terminal V5 epitope and His tag tail (encoding
SEQ ID NO: 37) and addition of a double stop codon. The construct (with and
without the tail) was then placed between the CaMV35s promoter and ocs
terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO: 110 and
SEQ ID NO: 116 respectively.
Plant Materials and Growth Conditions
Arabidopsis thaliana (L.)Heynh ecotype Ler can be used as wild-type (WT). The
Appd loss of function deletion mutant (with PPD1 and PPD2 deleted) is as
previously described in White 2006, PNAS 103, 13238-13243.
Plants are grown in a temperature-controlled glasshouse at a continuous 21°C or
in a controlled environment cabinet at 23°C in 16-h lig ht 8-h dark cycles.
Transformation of Arabidopsis
Constructs above can be transformed into Arabidopsis by the floral dip infiltration
method (Clough and Bent, 1998, Plant J 16, 735-43). The Appd line is
transformed to express the PPD polypeptides by standard techniques. Transgenic
plants are confirmed by standard PCR analysis techniques with a combination of
transgene-specific and T-DNA primers.
Complementation of the Appd line to produce a wild-type leaf and rosette
phenotype in Tl seedlings (the off-spring of the infiltrated plant) confirms
PEAPOD functionality of the introduced gene, which can be shown in photographs.
This approach can be use to confirm the PEAPOD functionality of any gene which
the applicant asserts, demonstrates it suitability of use in the present invention.
The PEAPOD proteins from Arabidopsis thaliana; Picea sitchensis, Amborella
trichopoda, Musa acuminate, and Selaginella moellendorffii were shown to be
functionally equivalent by the complementation of the PEAPOD deletion mutant
Appd Arabidopsis thaliana ecotype Landsberg erecta (Figure 13).

CLAIMS:
1. A method for increasing at least one of root biomass and above-ground
biomass and in a Poaceae plant, the method comprising the step of expressing a
PEAPOD protein, or fragment thereof, in the Poaceae plant.
2. The method of claim 1 in which the PEAPOD protein, or fragment thereof, is
expressed as a consequence of the plant, or its ancestor plant or plant cell,
having been transformed with a polynucleotide encoding the PEAPOD protein, or
fragment thereof.
3. The method of claim 1 or 2 in which the plant is transgenic for a polynucleotide
expressing the PEAPOD protein, or fragment thereof.
4. A method for producing a Poaceae plant with at least one of increased root
biomass and increased above-ground biomass, the method comprising the step of
expressing a PEAPOD protein, or fragment thereof, in the Poaceae plant.
5. The method of claim 4 in which the Poaceae plant is transformed with a
polynucleotide encoding the PEAPOD protein, or fragment thereof.
6. The method of claim 4 or 5 comprising the step of transforming the Poaceae
plant, or transforming a Poaceae plant cell which is regenerated into the Poaceae
plant, with a polynucleotide encoding the PEAPOD protein, or fragment thereof.
7. The method of claim 6 which includes the additional step of testing or
assessing the plant for at least one of increased root biomass and increased
above-ground biomass.
8. The method of claim any one of claims 1 to 7 in which the PEAPOD protein, or
fragment thereof, is a polypeptide comprising the sequence of at least one of the
sequences of SEQ ID NO: 28, 29, 31, 32, 34 and 35.
9. The method of claim any one of claims 1 to 8 in which the PEAPOD protein is a
polypeptide comprising a sequence with at least 70% identity to any one of SEQ
ID NO: 1 to 26.
10. The method of any one of claims 1 to 9 in which expression is increased by
introducing a polynucleotide encoding the PEAPOD protein, or fragment thereof,
into the plant cell or plant.
11. The method of claim 10 in which the polynucleotide comprises a sequence
with at least 70% identity to the coding sequence of any one of SEQ ID NO: 80 to
104 or a fragment thereof.
12. The method of claim 10 in which the polynucleotide comprises a sequence
with at least 70% identity to the sequence of any one of SEQ ID NO: 80 to 104 or
a fragment thereof.
13. The method of any one of claims 10 to 12 in which the polynucleotide, or
fragment thereof, is introduced into the plant as part of an expression construct.
14. The method of claim 13 in which the expression construct comprises a
promoter operatively linked to the polynucleotide or fragment thereof.
15. The method of claim 14 in which the promoter is capable of driving, or
drives, expression of the operatively linked polynucleotide or fragment thereof,
constitutively in all tissues of the plant.
16. The method of claim 14 in which the promoter is a tissue-preferred
promoter.
17. The method of claim 14 in which the promoter is capable of driving, or
drives, expression of the operatively linked polynucleotide, or a fragment thereof,
in the above-ground parts of the plant.
18. The method of claim 14 in which the promoter is capable of driving, or
drives, expression of the operatively linked polynucleotide, or a fragment thereof,
in the below ground tissues of the plant.
19. A Poaceae plant expressing a PEAPOD protein, or fragment thereof, that has
at least one of:
a) increased root biomass, and
b) increased above-ground biomass,
as a result of expressing the PEAPOD protein, or fragment thereof.
20. The Poaceae plant of claim 19 wherein the PEAPOD protein, or fragment
thereof, is expressed as a consequence of the plant, or its ancestor plant or plant
cell, having been transformed with a polynucleotide encoding the PEAPOD
protein, or fragment thereof.
21. The Poaceae plant of claim 19 or 20 that is transgenic for a polynucleotide
expressing the PEAPOD protein, or fragment thereof.
22. The Poaceae plant of claim 20 or 21 in which the polynucleotide or fragment
thereof is operatively linked polynucleotide to a tissue-preferred promoter.
23. The Poaceae plant of claim 22 in which the promoter is capable of driving, or
drives, expression of the operatively linked polynucleotide, or a fragment thereof,
in the above-ground parts of the plant.
24. The Poaceae plant of claim 22 in which the promoter is capable of driving, or
drives, expression of the operatively linked polynucleotide, or a fragment thereof,
in the below ground tissues of the plant.
25. A cell, part, propagule or progeny of the plant of any one of claims 19 to 24
that is transgenic for at least one of:
a) the polynucleotide, and
b) the polynucleotide and operatively linked promoter.