# Patent

## Title
Plant responses

## Abstract

The present invention relates to methods and uses for improving traits in plants which are important in the field of agriculture. In particular, the methods and uses of the invention use a plant Hsf to increase plant productivity, water use efficiency, drought or pathogen resistance.

## Background
This application is a U.S. national phase application under 35 U.S.C. §371 of international application No. PCT/GB2008/050186, filed Mar. 17, 2008, which claims the benefit of GB Application No. 0704984.4, filed Mar. 15, 2007.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The submitted ASCII copy was created on Jan. 7, 2010, modified on Sep. 25, 2011, and is 3,516 bytes in size.
FIELD OF THE INVENTION
The present invention relates to methods and uses for improving traits in plants which are important in the field of agriculture. In particular, the methods and uses of the invention can be employed to increase plant productivity, for example by improving the way in which plants make use of the water resources available to them or by conferring pathogen resistance.
BACKGROUND OF THE INVENTION
External conditions that adversely affect growth, development or productivity trigger a wide range of plant responses, such as altered gene expression, cellular metabolism and changes in growth rates and crop yields. There are two types of stress: biotic stress is imposed by other organisms, such as a pathogen, whereas abiotic stress arises from an excess or deficit in the physical or chemical environment, such as drought, salinity, high or low temperature or high light. Biotic and abiotic stresses can reduce average plant productivity by 65% to 87%, depending on the crop.
An example of biotic stress is pathogen infection. Plants have evolved defensive mechanisms, such as the induction of the expression of specific resistance genes upon infection. It is known that resistance is heritable and plant breeders have been breeding varieties of crop plants with disease resistance ever since. However, pests and pathogens have also developed ways to compromise plant resistance. Pathogens are adaptive by their ability to evolve strains that defeat the resistance genes deployed in crop plants by plant breeders. This has led to the need of continually updating and replacing varieties with different genes or combinations of genes for resistance in response to the ever-changing pathogen populations. Therefore, new ways of improving pathogen resistance are needed (Crute et al 1998, Cook et al 1996).
In addition to pathogen infection, plants are exposed to varying environmental conditions. One important factor in the development of plants and thus in agriculture is the availability of water. Water is essential for crop production because plants require water for growth and tissue expansion. Thus, the supply of fresh water is essential for all forms of agriculture, although the amount of water required varies greatly between different agricultural types and climatic regions.
There has been limited success with conventional breeding to improve the way in which plants use the water resources available. Genetic engineering is therefore considered an alternative. Several genes that regulate drought response have been identified in the model plant Arabidopsis. These are categorised as responsive to dehydration and early response to dehydration genes (Valliyodan et al 2006). One of the factors identified in regulating cold and drought stress responsive gene expression in Arabidopsis is a family of transcription factors termed DREB, which interact with a dehydration responsive element. Overexpression of DREB results in significant drought tolerance under water limited conditions. However, resistance to drought often compromises development of these transgenic plants under normal conditions. It has been shown that overexpression of DREB1/CBF and DREB2A driven by the CaMV 35S promoter causes growth retardation under normal conditions (Valliyodan et al 2006, Sakuma et al 2006, Qiang et al 2000). Thus, there has so far been no success in genetically modifying plants so that they show improved and more efficient use of water under normal non-drought conditions as well as under water deficit conditions.
Although increasing drought tolerance is desirable in the face of global warming, from an agricultural point of view, drought resistance is usually linked to low productivity, and is thus of limited use in agricultural production. Also, severe water deficits are generally rare in viable agriculture. Therefore, reducing the amount of water used per unit yield is now seen as the most promising way forward.
This is increasingly important due to the rising amounts of water which are used in agriculture and the changing climate. Globally, some 2.7×103 km3 of water were used in agriculture in 2000. It is estimated that the production of 1 kg of wheat requires 1 m3 of water, and 1 kg of rice requires at least 1.2 m3 of water. In the 15 countries of the EU in 2003, an area equivalent to 15.5% of the arable and permanent crop area was irrigated, and irrigation comprised over half of the total water consumption (EEA 2003). Even within the humid, temperate climate of England, 147 kha of outdoor crops were irrigated in 2001 (about 3% of the cropped area), using 131×106 m3 of water (Morison et al 2008; Rijsberman. 2004; Richards 2004; Food and Agriculture Organisation (FAO) 2003; Parry et al 2005.)
Thus, how to reduce agricultural water use and make water resources more sustainable is an increasingly urgent question. There is a need to develop crops that require less water to produce sufficient yield under normal conditions in addition to showing improved drought resistance. The amount of yield produced per unit water used is referred to as ‘water productivity’, a well known term in agriculture (Morison et al., 2008).
All eukaryotic organisms respond to an increase in the ambient temperature with the expression of a group of proteins known as heat shock proteins (HSPs). Key factors in the regulation of the expression of Hsp genes are the heat shock transcription factors (Hsfs) that act by binding to a highly conserved palindromic heat shock response sequence in the promoters of the target genes. In addition to mediating the response to heat stress, Hsfs are thought to be involved in cellular responses to oxidative stress, heavy metals and other stress responses (Panchuk et al 2002, Panikulangara et al 2004).
It is known that the basic structure of Hsfs and of their promoter recognition site is conserved throughout the eukaryotic kingdom (Kotak et al 2004, Miller and Mittler 2006). Hsfs have a modular structure with a highly conserved N-terminal DNA binding and a C-terminal activation domain. Other conserved domains include an oligomerisation domain, a nuclear localisation sequence and a nuclear export sequence. Thus, Hsfs are easily recognised by their conserved motifs essential for their function as transcription factors (Kotak et al 2004, Miller and Mittler 2006, Nover et al 2001).
Yeast and Drosophila contain only one Hsf gene, while vertebrates are thought to have three Hsf genes. In plants, Hsf genes have been identified in many species, for example maize, the model plant Arabidopsis thaliana (21 Hsfs), soybean (34 Hsfs), rice (23 Hsfs), barley, potato, tomato (18 Hsfs) and others. Hsfs within the plant kingdom are highly conserved and divided into three classes (A, B and C). For example, it has been found that a class of Hsfs in Arabidopsis is closely related to Hsf from rice and to Hsfs identified from ESTs in barley, potato, tomato and soy bean (Nover et al 2001 and Kotak et al 2004).
The invention is aimed at solving or at least mitigating the problems discussed above by introducing and expressing a gene sequence encoding a plant heat shock transcription factor.
SUMMARY OF THE INVENTION
The invention relates to methods and uses for improving a plant's tolerance to abiotic or biotic stress, not including heat stress. The method comprises introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant. In particular, the invention provides methods and uses for improving traits in plants which are important in the field of agriculture selected from the group comprising improved productivity, preferably growth or yield, water use efficiency, water productivity, drought tolerance or pathogen resistance.
In one aspect, the invention provides a method for improving plant productivity comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.
The invention also provides a method for improving water use efficiency in plants comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.
Furthermore, there is provided a method for conferring pathogen resistance in plants comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.
The invention also relates to uses of a plant Hsf in improving plant productivity, plant water use efficiency, water productivity, drought tolerance or pathogen resistance. In one embodiment, plant water use efficiency and water productivity are improved under normal, non drought conditions.
DETAILED DESCRIPTION
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature.
In a first aspect, the invention relates to a method for improving plant productivity comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf in said plant.
Plant productivity can be assessed by measuring plant growth or plant yield. Preferably, the term is used to describe an improvement in yield. This can be assessed by measuring seed yield, such as increased seed biomass or increased number of seeds. It can be improved by increasing water productivity.
According to the different aspects and embodiments of the invention, the plant into which a plant Hsf of plant origin is introduced may be any monocot or dicot plant.
A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Maivaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. In one embodiment, the plant is oilseed rape.
Also included are biofuel and bioenergy crops such as rape/canola, linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat sorghum, rye, onion, leek, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species.
Preferably, the plant into which a plant Hsf is introduced is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
Preferred plants are maize, wheat, rice, oilseed rape, sorghum, soybean, potato, tomato, barley, pea, bean, field bean, lettuce, broccoli or other vegetable brassicas or poplar.
The polynucleotide according to the different aspects and embodiments of the invention comprises or consists of a plant heat shock transcription factor gene, i.e. a plant Hsf. The term plant heat shock transcription factor gene or plant Hsf refers to a nucleic acid sequence which encodes a plant heat shock transcription factor. The Hsf gene can be from genomic DNA and therefore contain introns, a cDNA copy synthesised from the Hsf3 mRNA or could be a completely synthetic copy of the coding sequence made by assembly of chemically synthesised oligonucleotides. The plant heat shock transcription factor gene sequence can be isolated from a plant and inserted into a vector/expression cassette for transformation, for example by using an artificial plant chromosome.
Within the scope of the invention is also a derivative of a Hsf gene, such as a mutant/mutated gene, chimeric gene or gene shuffled variant. For example, the mutant gene may be modified so that the resulting protein is constitutively active and cannot be inhibited by other components of the Hsf signalling pathway. The derivative gene expressed a protein which is biologically active. It may have 80% or more sequence homology with the wild type gene. Thus, the methods and uses of the invention also relates to methods and uses employing a Hsf derivative.
The Hsf polynucleotide is a transgene that is introduced in the plant. This can be carried out by various methods as known in the field of plant genetic engineering, for example using transformation with Agrobacterium or particle bombardment.
The plant heat shock transcription factor gene may be an exogenous gene, such as one or more Arabidopsis Hsf, overexpressed in a different plant species. Alternatively, the plant Hsf may be an endogenous plant Hsf, i.e. a plant Hsf that is endogenous to the plant in which it is introduced and overexpressed.
In one embodiment of the different aspects of the invention, the exogenous plant Hsf may originate from any plant, for example a family or species listed above and expressed in a different plant species according to the invention. There is a structural high similarity between Hsfs in the plant kingdom. Plant Hsfs are conserved throughout the plant kingdom and can be identified due to their conserved domains. Plant Hsfs are divided into three groups A, B and C. Thus, according to the invention, the plant Hsf may be selected from group A, B or C. For example, the plant Hsf may be an Arabidopsis Hsf, a tomato Hsf, such as LpHsfA1, LpHsfA2, LpHsfA3 or LpHsfB1. Alternatively, the plant Hsf may be derived from rice, wheat, pea, maize, tobacco or any crop cereal. Non limiting examples of known Hsfs which can be used according to the invention are given in tables 1 and 2.
















OsHsfA1a
AK100430 Os03g0854500



OsHsfA2c
AK072391 Os10g0419300



OsHsfA2b
AK101824 Os07g0178600



OsHsfA2a
AK069579 ABF98829



OsHsfA1a2d
AK066844 Os03g0161900



OsHsfA2e
AK068660 Os03g0795900



OsHsfA3
AK101934 XM_466050



OsHsfA4b
AK109856 Os01g0749300



OsHsfA4d
AK100412 Os05g0530400



OsHsfA5
AK065643 Os02g0496100



OsHsfA7
AK064271 Os01g0571300



OsHsfA9
AK064271 Os03g0224700



OsHsfB1
AK101182 Os09g0456800



OsHsfB2a
Os04g0568700



OsHsfB2b
AK101700 Os08g0546800



OsHsfB2c
AK106525 Os09g0526600



OsHsfB4a
P0461F06.21



OsHsfB4b
AK063952 Os07g0640900



OsHsfB4c
Os09g0455200



OsHsfB4d
AK069479 ABF96133



OsHsfC1a
AK066316 Os01g0625300



OsHsfC1b
AK106488 Os01g0733200



OsHsfC2a
Os02g0232000



OsHsfC2b
Os06g0553100







Arabidopsis thaliana










AtHsfA1a
AT4G17750



AtHsfA1b
AT5G16820



AtHsfA1d
AT1G32330



AtHsfA1e
AT3G02990



AtHsfA2
AT2G26150



AtHsfA3
AT5G03720



AtHsfA4a
AT4G18880



AtHsfA4c
AT5G45710



AtHsfA5
AT4G13980



AtHsfA6a
AT5G43840



AtHsfA6b
AT3G22830



AtHsfA7a
AT3G51910



AtHsfA7b
AT3G63350



AtHsfA8
AT1G67970



AtHsfA9
AT5G54070



AtHsfB1
AT4G36990



AtHsfB2a
AT5G62020



AtHsfB2b
AT4G11660



AtHsfB3
AT2G41690



AtHsfB4
AT1G46264



AtHsfC1
AT3G24520







Tomato


Lycopersicon esculentum










LpHsfA1
X67600



LpHsfA2
X67601



LpHsfA3
AF208544



LpHsfB1
X55347










Homology searches of plant sequence databases such as the expressed sequence tag (EST) cDNA databases using the HSF3 derived amino acid sequence readily detects many highly significant homologies corresponding to HSFs in the query species. In the examples below the amino acid sequence of HSF3 was used to query the following databases with a significance value (E) of less than 1e−10. The values and hits, including the identification number are all shown in table 2 below.







TABLE 2







Soybean









gb|AW569256.1| si64g09.y1 Gm-r1030 Glycine max cDNA clone
290
4e−78


gb|AW569138.1| si63g09.y1 Gm-r1030 Glycine max cDNA clone
287
2e−77


gb|BM086093.1| sah35d07.y1 Gm-c1036 Glycine max cDNA clone
238,
1e−62


gb|CA938396.1| sav31h12.y1 Gm-c1048 Glycine max cDNA clone
213
5e−55


gb|BM521654.1| sak60e12.y1 Gm-c1036 Glycine max cDNA clone
211
2e−54


gb|BM527729.1| sal65b05.y1 Gm-c1061 Glycine max cDNA clone
209
5e−54


gb|AW395668.1| sg73g10.y1 Gm-c1007 Glycine max cDNA clone
201
2e−51


gb|CA801396.1| sau05b03.y2 Gm-c1062 Glycine max cDNA clone
195
1e−49


gb|AW508573.1| si33f01.y1 Gm-r1030 Glycine max cDNA clone
190
4e−48


gb|BI786160.1| sai33f04.y1 Gm-c1065 Glycine max cDNA clone
189
6e−48


gb|AW164509.1| se74f12.y1 Gm-c1023 Glycine max cDNA clone
166
7e−41


gb|BI471764.1| sae83d02.y3 Gm-c1065 Glycine max cDNA clone
162
1e−39


gb|BE611683.1| sq86g05.y1 Gm-c1049 Glycine max cDNA clone
160
4e−39


gb|AW132703.1| se09a08.y1 Gm-c1013 Glycine max cDNA clone
159
6e−39


gb|BE347442.1| sp38d02.y1 Gm-c1043 Glycine max cDNA clone
151
2e−36


gb|AW203851.1| sf38h11.y1 Gm-c1028 Glycine max cDNA clone
149
9e−36


gb|BG839442.1|Gm01_17a10_A, Gm01_AAFC_ECORC_Glycine_max_cold—
147
2e−35


gb|BG352891.1| sab92f08.y1 Gm-c1040 Glycine max cDNA clone
146
6e−35


gb|BG789771.1| sae55c03.y1 Gm-c1051 Glycine max cDNA clone
142
1e−33


gb|AW596493.1| sj13a09.y1 Gm-c1032 Glycine max cDNA clone
141
2e−33


gb|BM523618.1| sam86d01.y2 Gm-c1087 Glycine max cDNA clone
139
7e−33


gb|BM188104.1| saj84g05.y1 Gm-c1074 Glycine max cDNA clone
139
7e−33


gb|BM094717.1| saj19h06.y1 Gm-c1066 Glycine max cDNA clone
139
7e−33


gb|AI900223.1| sc02f05.y1 Gm-c1012 Glycine max cDNA clone
139
7e−33


gb|BM732569.1| sal78h07.y1 Gm-c1061 Glycine max cDNA clone
136
6e−32


gb|BI498205.1| sag17c01.y1 Gm-c1080 Glycine max cDNA clone
136
6e−32


gb|BU764266.1| sas54g03.y1 Gm-c1023 Glycine max cDNA clone
134
3e−31


gb|BQ094759.1| san51d12.y1 Gm-c1052 Glycine max cDNA clone
134
3e−31


gb|BI894096.1| sai60a12.y1 Gm-c1068 Glycine max cDNA clone
134
3e−31


gb|AW703969.1| sk14g08.y1 Gm-c1023 Glycine max cDNA clone
134
4e−31


gb|BM527450.1| sal62a04.y1 Gm-c1061 Glycine max cDNA clone
133
6e−31


gb|BG405291.1| sac50e11.y1 Gm-c1062 Glycine max cDNA clone
133
6e−31


gb|BG840046.1|, Gm01_08b12_F, Gm01_AAFC_ECORC_Glycine_max_cold_ . . .
132
1e−30


gb|CA936104.1| sav05g11.y1 Gm-c1048 Glycine max cDNA clone
130
3e−30


gb|BE346810.1| sp31e01.y1 Gm-c1042 Glycine max cDNA clone
130
3e−30


gb|BQ094171.1| san43b07.y1 Gm-c1052 Glycine max cDNA clone
130
4e−30


gb|BE020791.1| sm52h09.y1 Gm-c1028 Glycine max cDNA clone
127
3e−29


gb|CA850642.1| D04F08.seq cDNA Peking library 2, 4 day SCN3
126
6e−29


gb|BM886719.1| sam29c06.y1 Gm-c1068 Glycine max cDNA clone
126
6e−29


gb|BU577235.1| sar67d03.y1 Gm-c1074 Glycine max cDNA clone
126
8e−29


gb|BF071322.1| st45a08.y1 Gm-c1067 Glycine max cDNA clone
125
1e−28


gb|CX711571.1| gmrtDrNS01_35-D_M13R_F05_037.s2 Water stressed . . .
116
6e−26


gb|BQ474006.1| sap25b06.y1 Gm-c1082 Glycine max cDNA clone
115
1e−25


gb|CO984075.1| GM89021A1G02.r1 Gm-r1089 Glycine max cDNA
109
7e−24


gb|BE019974.1| sm38b12.y1 Gm-c1028 Glycine max cDNA clone
108
1e−23


gb|CX711887.1| gmrtDrNS01_39-D_M13R_C04_028.s3
84.7
5e−22


gb|BF067962.1| st79c06.y1 Gm-c1054 Glycine max cDNA clone
103
5e−22


gb|BU548776.1| GM880016B20F09 Gm-r1088 Glycine max cDNA
103
7e−22


gb|CA953210.1| sav53h02.y1 Gm-c1069 Glycine max cDNA clone
102
9e−22


gb|CA801977.1| sau28a12.y1 Gm-c1062 Glycine max cDNA clone
100
3e−21


gb|AW756148.1| sl16e07.y1 Gm-c1036 Glycine max cDNA clone
99.4
1e−20


gb|CD403874.1| Gm_ck26662 Soybean induced by Salicylic Acid G.
90.9
4e−18


gb|BE330669.1| so82h05.y1 Gm-c1040 Glycine max cDNA clone
89.4
1e−17


gb|BI469342.1| sai10f07.y1 Gm-c1053 Glycine max cDNA clone
89.0
1e−17


gb|BM271159.1| sak05h06.y1 Gm-c1074 Glycine max cDNA clone
87.0
5e−17


gb|DY577402.1| sgs2c.pk001.j19 DupontLib. Glycine max cDNA 5′, m
86.7
7e−17


gb|CX705290.1| gmrtDrNS01_40-B_M13R_H02_002.s2
85.9
1e−16


gb|AW508846.1| si41a12.y1 Gm-r1030 Glycine max cDNA clone GEN . . .
85.1
2e−16


gb|BU578607.1| sar59b05.y1 Gm-c1074 Glycine max cDNA clone
82.8
1e−15


gb|BQ473641.1| sap15g12.y1 Gm-c1082 Glycine max cDNA clone
82.0
2e−15


gb|BE348040.1| sp10e12.y1 Gm-c1042 Glycine max cDNA clone
79.7
8e−15


gb|BF425514.1| su56f05.y1 Gm-c1069 Glycine max cDNA clone
79.3
1e−14


gb|BQ628408.1| sap46f03.y1 Gm-c1087 Glycine max cDNA clone
79.0
1e−14


gb|BM269600.1| sak01h05.y1 Gm-c1074 Glycine max cDNA clone
76.6
7e−14


gb|AW620962.1| sj98b03.y1 Gm-c1023 Glycine max cDNA clone
76.6
7e−14


gb|AW704152.1| sk28b02.y1 Gm-c1028 Glycine max cDNA clone
75.1
2e−13


gb|BI316569.1| saf05a10.y1 Gm-c1065 Glycine max cDNA clone
74.3
3e−13


gb|BU760760.1| sas58b07.y1 Gm-c1023 Glycine max cDNA clone
72.8
1e−12


gb|BE475593.1| sp78e05.y1 Gm-c1044 Glycine max cDNA clone
68.2
3e−11







Barley









gb|BI951809.1| HVSMEm0003A03f Hordeum vulgare green seedling
241
2e−63


gb|DN182018.1| HO22J02S HO Hordeum vulgare cDNA clone HO22J02 . . .
239
1e−62


dbj|AV833112.1| AV833112 K. Sato unpublished cDNA library:
235
2e−61


dbj|AV941967.1| AV941967 K. Sato unpublished cDNA library
201
2e−51


gb|BU967095.1| HB03E12r BC Hordeum vulgare subsp. vulgare
193
6e−49


gb|BU967280.1| HB03N01r BC Hordeum vulgare subsp. vulgare
190
7e−48


gb|CA002527.1| HS07M12r HS Hordeum vulgare subsp. vulgare
177
4e−44


gb|BQ466839.1| HS01L22T HS Hordeum vulgare subsp. vulgare
177
4e−44


gb|BM373839.2| EBma03_SQ002_N22_R maternal, 8 DPA
176
1e−43


gb|BQ466452.1| HT02J04r HT Hordeum vulgare subsp. vulgare
176
1e−43


gb|BQ762325.1| EBro01_SQ005_B18 _R root, 3 week,
175
2e−43


gb|BF628773.2| HVSMEb0008B10f Hordeum vulgare seedling shoot
164
5e−40


gb|BF264338.2| HV_CEa0009C13f Hordeum vulgare seedling green
159
1e−38


gb|DN183502.1| HO17K20S HO Hordeum vulgare cDNA clone HO17K20 . . .
156
8e−38


gb|BQ466741.1| HS01H02T HS Hordeum vulgare subsp. vulgare
154
3e−37


gb|DN156902.1| GCN003J14u GCN Hordeum vulgare cDNA clone GCN0 . . .
154
5e−37


gb|BQ660530.1| HI04A10u HI Hordeum vulgare subsp. vulgare
152
1e−36


gb|CB870232.1| HC13L22w CH Hordeum vulgare cDNA clone HC13L22 . . .
152
2e−36


gb|BE216310.2| HV_CEb0010C13f Hordeum vulgare seedling green
151
3e−36


gb|BQ740081.1| HC04G06 HC Hordeum vulgare subsp. vulgare cDNA
150
6e−36


gb|BI959876.1| HVSMEn0022C22f Hordeum vulgare rachis EST
146
8e−35


gb|BQ660415.1| HI02G20u HI Hordeum vulgare subsp. vulgare
143
7e−34


gb|CA019131.1| HV10O05r HV Hordeum vulgare subsp. vulgare
138
3e−32


gb|CA028721.1| HZ63A24r HZ Hordeum vulgare subsp. vulgare
137
4e−32


gb|BU969351.1| HB11E12r BC Hordeum vulgare subsp. vulgare
136
9e−32


gb|BF616419.2| HVSMEc0007I04f Hordeum vulgare seedling shoot
134
6e−31


gb|BI948455.1| HVSMEI0009K13f Hordeum vulgare spike EST
133
1e−30


dbj|BY838837.1| BY838837 Etiolated seedling shoot Hordeum
132
2e−30


dbj|BY847793.1| BY847793 Seminal root Hordeum vulgare subsp.
132
2e−30


gb|CA003800.1| HS15J20r HS Hordeum vulgare subsp. vulgare
132
2e−30


gb|CA001818.1| HS05K18r HS Hordeum vulgare subsp. vulgare
132
2e−30


gb|BM369160.2| EBem07_SQ002_L22_R embryo, 28 DPA
132
2e−30


dbj|BY853068.1| BY853068 Germination shoots Hordeum vulgare
129
2e−29


dbj|BY847188.1| BY847188 Seminal root Hordeum vulgare subsp.
129
2e−29


gb|BF264951.3| HV_CEa0010N10f Hordeum vulgare seedling green . . .
128
3e−29


gb|BQ739839.1| HB04B12 HB Hordeum vulgare subsp. vulgare cDNA.
125
3e−28


gb|BQ758567.1| EBma07_SQ002_K03_R maternal, 21 DPA
124
6e−28


dbj|BY848814.1| BY848814 Seminal root Hordeum vulgare subsp.
121
4e−27


gb|DN183559.1| HO17C03S HO Hordeum vulgare cDNA clone HO17C03
95.1
1e−26


gb|CV063030.1| BNEL85h11 Barley EST endosperm library Hordeum
119
1e−26


gb|CV059324.1| BNEL46h12 Barley EST endosperm library Hordeum.
119
1e−26


gb|CV055928.1| BNEL12A4 Barley EST endosperm library Hordeum
119
1e−26


gb|BM098554.2| EBem08_SQ003_G12_R embryo, 40 DPA
117
7e−26


gb|BM098700.2| EBem08_SQ003_N07_R embryo, 40 DPA
116
1e−25


gb|CB873818.1| HC13L22y CH Hordeum vulgare cDNA clone HC13L22
115
2e−25


gb|BE602936.2| HVSMEh0101B12f Hordeum vulgare 5-45 DAP spike
115
3e−25


dbj|BY847001.1| BY847001 Seminal root Hordeum vulgare subsp.
93.2
6e−25


gb|CA001991.1| HS06D04r HS Hordeum vulgare subsp. vulgare
113
8e−25


gb|CA008447.1| HU10P22r HU Hordeum vulgare subsp. vulgare
112
1e−24


gb|CV057176.1| BNEL24h11 Barley EST endosperm library Hordeum.
111
3e−24


gb|BE216016.3| HV_CEb0009C04f Hordeum vulgare seedling green
77.8
9e−24


dbj|BY849120.1| BY849120 Germination shoots Hordeum vulgare
109
1e−23


gb|CV057937.1| BNEL32e5 Barley EST endosperm library Hordeum
107
6e−23


gb|BQ759041.1| EBma07_SQ003_H17_R maternal, 21 DPA
102
1e−21


dbj|BY849968.1| BY849968 Germination shoots Hordeum vulgare
102
2e−21


gb|BQ758691.1| EBma07_SQ002_D17_R maternal, 21 DPA
100
9e−21


dbj|BY850372.1| BY850372 Germination shoots Hordeum vulgare
90.9
6e−18


gb|BQ463252.1| HI04I11r HI Hordeum vulgare subsp. vulgare
90.5
7e−18


dbj|AV834862.1| AV834862 K. Sato unpublished cDNA library: Ho . . .
89.7
1e−17


gb|BU990279.1| HF24J17r HF Hordeum vulgare subsp. vulgare cDNA
88.6
3e−17


dbj|BY854029.1| BY854029 Germination shoots Hordeum vulgare
84.0
7e−16


gb|CA001926.1| HS06A02r HS Hordeum vulgare subsp. vulgare cDNA
84.0
7e−16


gb|BM815949.1| HB108G05_SK.ab1 HB Hordeum vulgare subsp.
84.0
7e−16


gb|BF619429.2| HVSMEc0003F06f Hordeum vulgare seedling shoot
84.0
7e−16


gb|BF630021.2| HVSMEb0007L02f Hordeum vulgare seedling shoot
84.0
7e−16


gb|BF265538.1| HV_CEa0012I16f Hordeum vulgare seedling green
82.0
3e−15


dbj|BJ477649.1| BJ477649 K. Sato unpublished cDNA library
77.0
8e−14


dbj|BJ469794.1| BJ469794 K. Sato unpublished cDNA library
68.9
2e−13


gb|BU997524.1| HI08D24r HI Hordeum vulgare subsp. vulgare
69.7
1e−11







Maize









gb|EE046500.1| ZM_BFc0116004.r ZM_BFc Zea mays cDNA clone
354
3e−97


gb|DR827048.1| ZM_BFb0070B11.f ZM_BFb Zea mays cDNA 3′, mRNA seq
320
7e−87


gb|EE153197.1| ZM_BFc0057D18.f ZM_BFc Zea mays cDNA clone
301
4e−81


gb|EE176144.1| ZM_BFc0155L14.f ZM_BFc Zea mays cDNA clone
300
1e−80


gb|EE016215.1| ZM_BFc0066O22.r ZM_BFc Zea mays cDNA clone
283
1e−75


gb|DR814130.1| ZM_BFb0043K05.r ZM_BFb Zea mays cDNA 5′, mRNA seq
251
3e−66


gb|EE044700.1| ZM_BFc0113I19.r ZM_BFc Zea mays cDNA clone
237
8e−62


gb|CO466103.1| MZCCL20041E02.g Maize Endosperm cDNA Library
228
3e−59


gb|CX129539.1| QCD4f05.yg QCD Zea mays cDNA clone QCD4f05, mRNA
223
2e−57


gb|EE020758.1| ZM_BFc0074B16.r ZM_BFc Zea mays cDNA clone
213
1e−54


gb|DR830042.1| ZM_BFb0077D11.r ZM_BFb Zea mays cDNA 5′, mRNA seq
213
1e−54


gb|EE036952.1| ZM_BFc0100H21.r ZM_BFc Zea mays cDNA clone
205
3e−52


gb|EE174627.1| ZM_BFc0153H07.r ZM_BFc Zea mays cDNA clone
200
1e−50


gb|EE168641.1| ZM_BFc0143L05.r ZM_BFc Zea mays cDNA clone
200
1e−50


gb|EC878082.1| ZM_BFc0012B11.r ZM_BFc Zea mays cDNA clone
200
1e−50


gb|DV531708.1| ZM_BFb0221P10.r ZM_BFb Zea mays cDNA 5′, mRNA seq
200
1e−50


gb|DR819313.1| ZM_BFb0055J22.r ZM_BFb Zea mays cDNA 5′, mRNA seq
200
1e−50


gb|DR814313.1| ZM_BFb0043O15.r ZM_BFb Zea mays cDNA 5′, mRNA seq
200
1e−50


gb|DV521145.1| ZM_BFb0206F11.r ZM_BFb Zea mays cDNA 5′, mRNA seq
199
2e−50


gb|EE023205.1| ZM_BFc0078F16.r ZM_BFc Zea mays cDNA clone
197
5e−50


gb|DT644796.1| ZM_BFb0104H15.r ZM_BFb Zea mays cDNA 5′, mRNA seq
197
5e−50


gb|EE045614.1| ZM_BFc0115D04.r ZM_BFc Zea mays cDNA clone
129
6e−50


gb|EE184383.1| ZM_BFc0168M02.r ZM_BFc Zea mays cDNA clone
197
7e−50


gb|EB676377.1| ZM_BFb0340P21.r ZM_BFb Zea mays cDNA 5′, mRNA seq
197
9e−50


gb|EB641134.1| ZM_BFb0330I04.r ZM_BFb Zea mays cDNA 5′, mRNA seq
197
9e−50


gb|DV514179.1| ZM_BFb0196A08.r ZM_BFb Zea mays cDNA 5′, mRNA seq
197
9e−50


gb|DT938818.1| ZM_BFb0120H03.r ZM_BFb Zea mays cDNA 5′, mRNA seq
197
9e−50


gb|EC878282.1| ZM_BFc0012G03.r ZM_BFc Zea mays cDNA clone
194
6e−49


gb|DR803312.1| ZM_BFb0027O21.r ZM_BFb Zea mays cDNA 5′, mRNA seq
192
2e−48


gb|DV536183.1| ZM_BFb0228J07.r ZM_BFb Zea mays cDNA 5′, mRNA seq
192
3e−48


gb|EE016587.1| ZM_BFc0067H20.r ZM_BFc Zea mays cDNA clone
110
1e−47


gb|EE037596.1| ZM_BFc0101H15.r ZM_BFc Zea mays cDNA clone
188
3e−47


gb|EE181056.1| ZM_BFc0163K15.r ZM_BFc Zea mays cDNA clone
186
2e−46


gb|EB821749.1| ZM_BFb0383A18.r ZM_BFb Zea mays cDNA 5′, mRNA seq
184
5e−46


gb|DR972034.1| ZM_BFb0095A05.r ZM_BFb Zea mays cDNA 5′, mRNA seq
182
3e−45


gb|EE046747.1| ZM_BFc0119D19.r ZM_BFc Zea mays cDNA clone
179
2e−44


gb|EC898631.1| ZM_BFc0043J13.r ZM_BFc Zea mays cDNA clone
100
2e−41


gb|EE016207.1| ZM_BFc0066O16.r ZM_BFc Zea mays cDNA clone
166
2e−40


gb|EE172761.1| ZM_BFc0150H22.r ZM_BFc Zea mays cDNA clone
97.1
3e−40


gb|EC893479.1| ZM_BFc0035L05.r ZM_BFc Zea mays cDNA clone
165
4e−40


gb|CA828716.1| 1114032E05.y2 1114 - Unigene IV from Maize
164
7e−40


gb|DY689425.1| ZM_BFb0285C17.r ZM_BFb Zea mays cDNA 5′, mRNA seq
162
2e−39


gb|DV538370.1| ZM_BFb0231M05.r ZM_BFb Zea mays cDNA 5′, mRNA seq
162
2e−39


gb|DR965723.1| ZM_BFb0085M05.r ZM_BFb Zea mays cDNA 5′, mRNA seq
162
2e−39


gb|DV167013.1| ZM_BFb0164N15.r ZM_BFb Zea mays cDNA 5′, mRNA seq
161
4e−39


gb|DR820230.1| ZM_BFb0058D10.r ZM_BFb Zea mays cDNA 5′, mRNA seq
161
4e−39


gb|BU079565.1| 946145D05.y1 946 - tassel primordium prepared
161
4e−39


gb|EE165047.1| ZM_BFc0137E21.r ZM_BFc Zea mays cDNA clone
161
6e−39


gb|EE037488.1| ZM_BFc0101E17.r ZM_BFc Zea mays cDNA clone
160
7e−39


gb|DV519877.1| ZM_BFb0204H22.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DV508968.1| ZM_BFb0187M19.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DV168574.1| ZM_BFb0167E09.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DV033268.1| ZM_BFb0157G21.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DV019914.1| ZM_BFb0138A11.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DT641438.1| ZM_BFb0099I01.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DR970291.1| ZM_BFb0092H08.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DR969885.1| ZM_BFb0091N01.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|DR828268.1| ZM_BFb0072L05.r ZM_BFb Zea mays cDNA 5′, mRNA seq
160
7e−39


gb|EE026988.1| ZM_BFc0084J01.r ZM_BFc Zea mays cDNA clone ZM_ . . .
160
9e−39


gb|DN560588.1| ME24-A03-T3-96-R1 E7PCR Zea mays cDNA clone E7 . . .
160
1e−38


gb|BM500210.1| PAC000000000320 Pioneer AF-1 array Zea mays cDNA,
160
1e−38


gb|DV032779.1| ZM_BFb0156L04.r ZM_BFb Zea mays cDNA 5′, mRNA seq
159
2e−38


gb|DR828476.1| ZM_BFb0073E20.r ZM_BFb Zea mays cDNA 5′, mRNA seq
159
2e−38


gb|CA830206.1| 1117003H06.y1 1117 - Unigene V from Maize
159
2e−38


gb|EC891458.1| ZM_BFc0032K08.r ZM_BFc Zea mays cDNA clone
158
4e−38


gb|EC879486.1| ZM_BFc0014C20.r ZM_BFc Zea mays cDNA clone
158
4e−38


gb|CO451095.1| MZCCL10160C11.g Maize Endosperm cDNA Library
90.1
7e−38


gb|BQ295716.1| 1091041H03.y1 1091
157
8e−38


gb|CO525958.1| 3530_1_172_1_F12.y_1 3530 - Full length cDNA
154
7e−37


gb|CO466022.1| MZCCL20042D10.g Maize Endosperm cDNA Library
154
9e−37


gb|DY240393.1| ZM_BFb0259N07.r ZM_BFb Zea mays cDNA 5′, mRNA seq
153
1e−36


gb|EE044234.1| ZM_BFc0112N15.r ZM_BFc Zea mays cDNA clone
152
3e−36


gb|EE020950.1| ZM_BFc0074H05.r ZM_BFc Zea mays cDNA clone
150
1e−35


gb|DR807887.1| ZM_BFb0034F24.r ZM_BFb Zea mays cDNA 5′, mRNA seq
150
1e−35


gb|CF002965.1| QBH17e07.xg QBH Zea mays cDNA clone QBH17e07, mRN
147
6e−35


gb|EC886519.1| ZM_BFc0025E04.r ZM_BFc Zea mays cDNA clone
147
1e−34


gb|CO518740.1| 3530_1_122_1_E05.y_1 3530 - Full length cDNA
146
1e−34


gb|CF244820.1| 3530_1_5_1_A07.y_2 3530 - Full length cDNA
146
1e−34


gb|EE015293.1| ZM_BFc0064K18.r ZM_BFc Zea mays cDNA clone
144
7e−34


gb|CF629555.1| zmrws48_0A20-002-g05.s0 zmrws48 Zea mays cDNA 3′,
140
8e−33


gb|CD443521.1| EL01N0427E10.b Endosperm_4 Zea mays cDNA, mRNA se
140
8e−33


gb|EE013968.1| ZM_BFc0062H12.r ZM_BFc Zea mays cDNA clone
140
1e−32


gb|EE013967.1| ZM_BFc0062H12.f ZM_BFc Zea mays cDNA clone
140
1e−32


gb|DR967139.1| ZM_BFb0087N19.r ZM_BFb Zea mays cDNA 5′, mRNA seq
140
1e−32


gb|AW065936.1| 687003D06.y1 687
140
1e−32


gb|BQ295608.1| 1091038B11.y1 1091
140
1e−32


gb|EC880371.1| ZM_BFc0015I01.r ZM_BFc Zea mays cDNA clone
139
3e−32


gb|DY688753.1| ZM_BFb0284B21.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DY624757.1| ZM_BFb0347H01.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV539151.1| ZM_BFb0232N24.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV537032.1| ZM_BFb0229M13.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV535973.1| ZM_BFb0228E13.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV535514.1| ZM_BFb0227J20.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV530458.1| ZM_BFb0220E01.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV515662.1| ZM_BFb0198F14.r ZM_BFb Zea mays cDNA5′, mRNA seq
139
3e−32


gb|DV507107.1| ZM_BFb0185C03.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV025493.1| ZM_BFb0146B22.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DV025290.1| ZM_BFb0145N07.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DT946133.1| ZM_BFb0133K13.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32


gb|DT645771.1| ZM_BFb0105P04.r ZM_BFb Zea mays cDNA 5′, mRNA seq
139
3e−32







Wheat









gb|DR735777.1| FGAS081411 Triticum aestivum
319
2e−86


dbj|CJ649060.1| CJ649060 Y. Ogihara unpublished cDNA library
238
4e−62


gb|DR740109.1| FGAS085037 Triticum aestivum FGAS: Library
197
9e−50


gb|CD881399.1| F1.103B17F010329 F1 Triticum aestivum cDNA
194
6e−49


dbj|CJ655373.1| CJ655373 Y. Ogihara unpublished cDNA library
189
2e−47


gb|CD884036.1| F1.115E10F010507 F1 Triticum aestivum cDNA
186
2e−46


gb|CV762533.1| FGAS056922 Triticum aestivum FGAS: Library 2
185
5e−46


gb|DR740033.1| FGAS084961 Triticum aestivum FGAS: Library 2
184
6e−46


gb|BI479783.1| WHE3452_A08_A16ZS Wheat pre-anthesis spike cDNA.
175
5e−43


gb|CD919573.1| G608.113N06F010911 G608 Triticum aestivum cDNA
164
7e−40


gb|CD939626.1| OV.114E09F010312 OV Triticum aestivum cDNA
162
4e−39


emb|AL812570.1| AL812570 e: 310 Triticum aestivum cDNA clone
157
1e−37


gb|CD871372.1| AZO2.118B14F010207 AZO2 Triticum aestivum cDNA . . .
157
1e−37


gb|CD909725.1| G468.113G04F010820 G468 Triticum aestivum cDNA
154
7e−37


dbj|BJ304701.1| BJ304701 Y. Ogihara unpublished cDNA library
152
3e−36


gb|CV766170.1| FGAS060557 Triticum aestivum FGAS: Library 2
152
3e−36


gb|BE488911.1| WHE1077_G04_N07ZS Wheat unstressed seedling
152
4e−36


gb|BE418746.1| SCL074.E11R990812 ITEC SCL Wheat Leaf Library
151
8e−36


gb|CD868305.1| AZO2.108I04F001113 AZO2 Triticum aestivum cDNA.
148
5e−35


gb|CD918044.1| G608.107M05F010905 G608 Triticum aestivum cDNA.
147
1e−34


gb|CA733194.1| wlp1c.pk007.b24 wlp1c Triticum aestivum cDNA
147
1e−34


gb|CK208279.1| FGAS019979 Triticum aestivum FGAS: Library 5
146
2e−34


dbj|CJ627951.1| CJ627951 Y. Ogihara unpublished cDNA library
145
4e−34


gb|CK213161.1| FGAS025066 Triticum aestivum FGAS: Library 6
143
2e−33


gb|CV781279.1| FGAS075690 Triticum aestivum FGAS: Library 2
137
9e−32


gb|CK196713.1| FGAS005173 Triticum aestivum FGAS: Library 3
137
1e−31


gb|CD894087.1| G118.125F14F010828 G118 Triticum aestivum cDNA
137
1e−31


gb|CA741191.1| wia1c.pk001.g8 wia1c Triticum aestivum cDNA
137
1e−31


gb|BQ240791.1| TaE05012H01R TaE05 Triticum aestivum cDNA
137
1e−31


gb|BU100082.1| WHE3314_H10_O20ZS Chinese Spring wheat drought
137
1e−31


emb|AJ601747.1| AJ601747 T05 Triticum aestivum cDNA clone
135
3e−31


gb|BU100475.1| WHE3353_G05_N09ZS Chinese Spring aluminum-stre . . .
135
3e−31


gb|BE426590.1| WHE0336_F08_L16ZS Wheat unstressed seedling
135
4e−31


gb|CD924507.1| G750.113E16F010706 G750 Triticum aestivum cDNA
135
6e−31


gb|BF200512.1| WHE0825-0828_B15_B15ZS
134
1e−30


gb|CV762050.1| FGAS056439 Triticum aestivum FGAS: Library
132
4e−30


gb|CD868306.1| AZO2.108I04R010328 AZO2 Triticum aestivum cDNA
132
4e−30


gb|CD924855.1| G750.114O18F010706 G750 Triticum aestivum cDNA
131
6e−30


gb|BE471094.1| WHE0284_H12_O24ZS
130
1e−29


gb|CV780245.1| FGAS074654 Triticum aestivum FGAS: Library 2
128
5e−29


emb|AJ602329.1| AJ602329 T05 Triticum aestivum cDNA clone
127
9e−29


gb|CV768109.1| FGAS062500 Triticum aestivum FGAS: Library 2
127
2e−28


gb|CD881419.1| F1.103C19F010328 F1 Triticum aestivum cDNA
125
3e−28


gb|CD920551.1| G608.117J13F010912 G608 Triticum aestivum cDNA
125
4e−28


dbj|CJ660695.1| CJ660695 Y. Ogihara unpublished cDNA library
123
2e−27


dbj|CJ696176.1| CJ696176 Y. Ogihara unpublished cDNA library
122
3e−27


gb|DR736601.1| FGAS081971 Triticum aestivum FGAS: Library 5
120
2e−26


dbj|CJ627959.1| CJ627959 Y. Ogihara unpublished cDNA library
119
2e−26


dbj|CJ501729.1| CJ501729 Y. Ogihara unpublished cDNA library
118
5e−26


gb|CV780793.1| FGAS075204 Triticum aestivum FGAS: Library 2
118
7e−26


gb|CA701457.1| wkm2c.pk006.e15 wkm2c Triticum aestivum cDNA
105
8e−26


gb|CV775774.1| FGAS070178 Triticum aestivum FGAS: Library 2
102
3e−25


gb|CD919574.1| G608.113N06R011027 G608 Triticum aestivum cDNA.
115
4e−25


gb|BE499216.1| WHE0972_H11_O22ZS Wheat pre-anthesis spike cDNA . . .
112
4e−24


gb|CK216192.1| FGAS028177 Triticum aestivum FGAS: Library 6
110
1e−23


gb|CA632168.1| wle1n.pk0062.f9 wle1n Triticum aestivum cDNA
110
1e−23


emb|AL816629.1| AL816629 I: 226 Triticum aestivum cDNA clone
67.4
4e−23


gb|CV758782.1| FGAS053164 Triticum aestivum FGAS: Library 2
107
1e−22


gb|BE406522.1| WHE0417_f11_k21zB
106
3e−22


gb|CA732800.1| wlp1c.pk004.j3 wlp1c Triticum aestivum cDNA
103
2e−21


gb|CA697753.1| wlk4.pk0010.g3 wlk4 Triticum aestivum cDNA
61.6
5e−21


gb|CD882214.1| F1.105L19F010330 F1 Triticum aestivum cDNA
101
7e−21


gb|CA657309.1| wlm0.pk0034.e2 wlm0 Triticum aestivum cDNA
97.8
1e−19


dbj|CJ712793.1| CJ712793 Y. Ogihara unpublished cDNA library
93.6
2e−18


dbj|CJ519157.1| CJ519157 Y. Ogihara unpublished cDNA library
92.0
5e−18


dbj|CJ712794.1| CJ712794 Y. Ogihara unpublished cDNA library
91.7
7e−18


gb|CD918480.1| G608.109J07F010907 G608 Triticum aestivum cDNA
91.7
7e−18


gb|BQ839307.1| WHE4164_F06_K12ZS Wheat CS whole plant cDNA
90.1
2e−17


emb|AL818825.1| AL818825 I: 125 Triticum aestivum cDNA clone
87.0
2e−16


gb|BF202987.1| WHE1768_A07_A14ZS Wheat pre-anthesis spike cDNA . . .
84.7
9e−16


gb|CD925960.1| G750.119H10F010711 G750 Triticum aestivum cDNA . . .
84.0
1e−15


dbj|CJ608117.1| CJ608117 Y. Ogihara unpublished cDNA library
82.4
4e−15


emb|AJ602330.1| AJ602330 T05 Triticum aestivum cDNA clone E08 . . .
82.4
4e−15


gb|CK211694.1| FGAS023548 Triticum aestivum FGAS: Library 6
81.6
7e−15


gb|BE470775.1| WHE0281_A11_B22ZS
81.6
7e−15


gb|CA610399.1| wr1.pk0119.f3 wr1 Triticum aestivum cDNA
65.9
9e−15


gb|DY741996.1| EST0565 Cold treated wheat cDNA library
81.3
1e−14


gb|CA596291.1| wpa1c.pk012.m23 wpa1c Triticum aestivum cDNA
81.3
1e−14


gb|BE427506.1| PSR7104 ITEC PSR Wheat Pericarp/Testa
76.6
1e−14


dbj|CJ640745.1| CJ640745 Y. Ogihara unpublished cDNA library
77.8
1e−13


gb|CA620714.1| wl1n.pk0067.b9 wl1n Triticum aestivum cDNA
75.9
4e−13


emb|AJ603615.1| AJ603615 T07 Triticum aestivum cDNA clone
75.5
5e−13


dbj|BJ232988.1| BJ232988 Y. Ogihara unpublished cDNA library
72.4
4e−12


gb|CA645001.1| wre1n.pk0086.b5 wre1n Triticum aestivum cDNA
71.2
1e−11


gb|CA484478.1| WHE4307_A11_B21ZS Wheat meiotic anther cDNA
67.0
2e−10







Potato









gb|CK253280.1| EST736917 potato callus cDNA library, normaliz . . .
324
2e−88


gb|CK267715.1| EST713793 potato abiotic stress cDNA library S . . .
323
3e−88


gb|CK246340.1| EST729977 potato callus cDNA library, normaliz . . .
308
1e−83


gb|CK244536.1| EST728173 potato callus cDNA library, normaliz . . .
306
6e−83


gb|BG890899.1| EST516750 cSTD Solanum tuberosum cDNA clone cS . . .
304
2e−82


gb|CK246138.1| EST729775 potato callus cDNA library, normaliz . . .
303
3e−82


gb|CK245930.1| EST729567 potato callus cDNA library, normaliz . . .
295
2e−80


gb|CK277468.1| EST723546 potato abiotic stress cDNA library S . . .
292
7e−79


gb|CK253321.1| EST736958 potato callus cDNA library, normaliz . . .
246
2e−76


gb|CK251625.1| EST735262 potato callus cDNA library, normaliz . . .
267
3e−71


gb|CK251428.1| EST735065 potato callus cDNA library, normaliz . . .
263
5e−70


gb|BQ510589.2| EST618004 Generation of a set of potato cDNA c . . .
243
5e−64


gb|DN922927.1| 44403.2 Common Scab-Challenged Tubers Solanum . . .
219
8e−57


gb|BG599468.1| EST504363 cSTS Solanum tuberosum cDNA clone cS . . .
214
3e−55


gb|CK269104.1| EST715182 potato abiotic stress cDNA library S . . .
212
1e−54


gb|CK262462.1| EST708540 potato abiotic stress cDNA library S . . .
212
1e−54


gb|CK252508.1| EST736145 potato callus cDNA library, normaliz . . .
212
1e−54


gb|CK245866.1| EST729503 potato callus cDNA library, normaliz . . .
212
1e−54


gb|CV429240.1| 51723.1 After-Cooking Darkening C Solanum tube . . .
211
2e−54


gb|BG890138.1| EST515989 cSTD Solanum tuberosum cDNA clone cS . . .
211
2e−54


gb|CV431358.1| 55572.1 After-Cooking Darkening C Solanum tube . . .
209
1e−53


gb|BF459947.1| 068G10 Mature tuber lambda ZAP Solanum tuberos . . .
205
1e−52


gb|CK264318.1| EST710396 potato abiotic stress cDNA library S . . .
204
3e−52


gb|BG594178.1| EST492856 cSTS Solanum tuberosum cDNA clone cS . . .
203
4e−52


gb|BI406849.1| 182A06 Mature tuber lambda ZAP Solanum tuberos . . .
201
2e−51


gb|BG595575.1| EST494253 cSTS Solanum tuberosum cDNA clone cS . . .
200
5e−51


gb|CK719979.1| 20306 Swollen Stolon Solanum tuberosum cDNA, mRNA
198
1e−50


gb|BG886998.1| EST512849 cSTD Solanum tuberosum cDNA clone cS . . .
198
1e−50


gb|BG890352.1| EST516203 cSTD Solanum tuberosum cDNA clone cS . . .
195
2e−49


gb|CN213018.1| 26561 Suspension culture Solanum tuberosum cDNA,
194
3e−49


gb|EG012052.1| STDB004A01u STDB Solanum tuberosum cDNA clone . . .
194
4e−49


gb|BG596222.1| EST494900 cSTS Solanum tuberosum cDNA clone cS . . .
168
5e−49


gb|CV474156.1| 22487.1 Developing Tubers Solanum tuberosum cD . . .
192
1e−48


gb|BI176643.1| EST517588 cSTE Solanum tuberosum cDNA clone cS . . .
190
4e−48


gb|BG890868.1| EST516719 cSTD Solanum tuberosum cDNA clone cS . . .
190
5e−48


gb|BE922055.1| EST425824 potato leaves and petioles Solanum t . . .
189
9e−48


gb|DN923069.1| 44928.2 Common Scab-Challenged Tubers Solanum . . .
188
1e−47


gb|BG887370.1| EST513221 cSTD Solanum tuberosum cDNA clone cS . . .
187
4e−47


gb|BG351853.1| 135A04 Mature tuber lambda ZAP Solanum tuberos . . .
186
7e−47


gb|BG591987.1| EST499829 P. infestans-challenged leaf Solanum . . .
180
4e−45


gb|BG889138.1| EST514989 cSTD Solanum tuberosum cDNA clone cS . . .
180
5e−45


gb|CK277133.1| EST723211 potato abiotic stress cDNA library S . . .
179
9e−45


gb|CK269720.1| EST715798 potato abiotic stress cDNA library S . . .
179
9e−45


gb|CK851489.1| 11654 Stolon Solanum tuberosum cDNA, mRNA sequenc
119
1e−44


gb|CN216247.1| 30125 Suspension culture Solanum tuberosum cDNA,
178
2e−44


gb|CK256967.1| EST740604 potato callus cDNA library, normaliz . . .
178
2e−44


gb|CK255235.1| EST738872 potato callus cDNA library, normaliz . . .
178
2e−44


gb|CK249408.1| EST733045 potato callus cDNA library, normaliz . . .
178
2e−44


gb|CK261460.1| EST707538 potato abiotic stress cDNA library S . . .
177
4e−44


gb|BQ121678.2| EST607254 mixed potato tissues Solanum tuberos . . .
176
6e−44


gb|BG886969.1| EST512820 cSTD Solanum tuberosum cDNA clone cS . . .
175
2e−43


gb|BG888694.1| EST514545 cSTD Solanum tuberosum cDNA clone cS . . .
172
1e−42


gb|BF459641.1| 062F02 Mature tuber lambda ZAP Solanum tuberos . . .
172
1e−42


gb|BF153546.1| 028C10 Mature tuber lambda ZAP Solanum tuberos . . .
172
1e−42


gb|BG597894.1| EST496572 cSTS Solanum tuberosum cDNA clone cS . . .
171
3e−42


gb|BG595042.1| EST493720 cSTS Solanum tuberosum cDNA clone cS . . .
169
7e−42


gb|CV430232.1| 53385.1 After-Cooking Darkening C Solanum tube . . .
169
9e−42


gb|BG890527.1| EST516378 cSTD Solanum tuberosum cDNA clone cS . . .
168
2e−41


gb|CV471720.1| 44928.1 Common Scab-Challenged Tubers Solanum . . .
167
4e−41


gb|BQ116126.2| EST601702 mixed potato tissues Solanum tuberos . . .
97.4
1e−40


gb|BQ511608.2| EST619023 Generation of a set of potato cDNA c . . .
163
5e−40


gb|CV496124.1| 73841.1 Cold Sweetening B Solanum tuberosum cD . . .
163
7e−40


gb|CK720145.1| 20511 Swollen Stolon Solanum tuberosum cDNA, mRNA
162
1e−39


gb|BI176663.1| EST517608 cSTE Solanum tuberosum cDNA clone cS . . .
161
2e−39


gb|BG888216.1| EST514067 cSTD Solanum tuberosum cDNA clone cS . . .
160
3e−39


gb|CK250720.1| EST734357 potato callus cDNA library, normaliz . . .
160
4e−39


gb|BG886541.1| EST512392 cSTD Solanum tuberosum cDNA clone cS . . .
160
6e−39


gb|CV471787.1| 45004.1 Common Scab-Challenged Tubers Solanum . . .
159
7e−39


gb|BI405991.1| 150C03 Mature tuber lambda ZAP Solanum tuberos . . .
159
1e−38


gb|DV623092.1| 92505.1 Cold Sweetening C Solanum tuberosum cD . . .
155
1e−37


gb|BE342382.1| EST395226 potato stolon, Cornell University So . . .
152
2e−36


gb|BI178192.1| EST519137 cSTE Solanum tuberosum cDNA clone cS . . .
151
2e−36


gb|BQ509197.2| EST616612 Generation of a set of potato cDNA c . . .
150
3e−36


gb|AW906822.1| EST342945 potato stolon, Cornell University So . . .
150
4e−36


gb|DN941337.1| 55572.2 After-Cooking Darkening C Solanum tube . . .
150
6e−36


gb|CN216727.1| 30658 Suspension culture Solanum tuberosum cDNA,
149
1e−35


gb|CK261481.1| EST707559 potato abiotic stress cDNA library S . . .
146
6e−35


gb|BQ510562.2| EST617977 Generation of a set of potato cDNA c . . .
146
8e−35


gb|CK851608.1| 11803 Stolon Solanum tuberosum cDNA, mRNA sequenc
145
1e−34


gb|BQ505416.2| EST612831 Generation of a set of potato cDNA c . . .
145
1e−34


gb|BG593665.1| EST492343 cSTS Solanum tuberosum cDNA done cS . . .
145
1e−34


gb|DN909107.1| 57843.2 Developing Tubers Solanum tuberosum cD . . .
145
2e−34


gb|DN907365.1| 22487.2 Developing Tubers Solanum tuberosum cD . . .
145
2e−34


gb|CV477893.1| 57843.1 Developing Tubers Solanum tuberosum cD . . .
145
2e−34


gb|CK274767.1| EST720845 potato abiotic stress cDNA library S . . .
143
5e−34


gb|CK258841.1| EST742478 potato callus cDNA library, normaliz . . .
143
5e−34


gb|CK258748.1| EST742385 potato callus cDNA library, normaliz . . .
143
7e−34


gb|BE920995.1| EST424764 potato leaves and petioles Solanum t . . .
142
9e−34


gb|DN923089.1| 45004.2 Common Scab-Challenged Tubers Solanum . . .
142
1e−33


gb|BF187134.1| EST443421 potato stolon, Cornell University So . . .
141
3e−33


gb|BF187133.1| EST443420 potato stolon, Cornell University So . . .
141
3e−33


gb|CK263459.1| EST709537 potato abiotic stress cDNA library S . . .
140
4e−33


gb|AW906840.1| EST342963 potato stolon, Cornell University So . . .
139
1e−32


gb|CK256905.1| EST740542 potato callus cDNA library, normaliz . . .
139
1e−32


gb|CV434757.1| 58247.1 Suspension culture Solanum tuberosum c . . .
137
3e−32


gb|CK278836.1| EST724914 potato abiotic stress cDNA library S . . .
137
3e−32


gb|BF052865.1| EST438095 potato leaves and petioles Solanum t . . .
137
3e−32


gb|BE471540.1| EST416393 potato stolon, Cornell University So . . .
134
3e−31


gb|DV625610.1| 95977.1 Cold Sweetening C Solanum tuberosum cD . . .
132
1e−30


gb|DN849475.1| 13215.2 Stolon Solanum tuberosum cDNA clone 13 . . .
132
1e−30









In one embodiment of the different aspects of the invention, an Arabidopsis Hsf selected from the Arabidopsis Hsf listed above, is overexpressed in another plant. The Arabidopsis Hsf is selected from the group comprising AtHsfA1a, AtHsfA1b, AtHsfA1d, AtHsfA1e, AtHsfA2, AtHsfA3, AtHsfA4a, AtHsfA4c, AtHsfA5, AtHsfA6a, AtHsfA6b, AtHsfA7a, AtHsfA7b, AtHsfA8, AtHsfA9, AtHsfB1, AtHsfB2a, AtHsfB2b, AtHsfB3, AtHsfB4 or AtHsfC1. In one embodiment, the Arabidopsis Hsf is AtHSFA1b. The full sequence of AtHSFA1b is shown in FIG. 1 (SEQ No 1).
The plant in which the Hsf is overexpressed may be any plant as listed herein. Preferably, the Arabidopsis Hsf, for example AtHSFA1b is overexpressed in a crop, for example a cereal, such as wheat, rice, barley, maize, oat sorghum, rye, onion, leek, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species. However, the applicability of the invention is not limited to the sequence shown in Seq ID No 1 as a skilled person would understand that other Hsfs isolated or derived from Arabidopsis or from other plants can also be used. Any combination of a plant Hsf for example as listed herein, in another plant, for example as listed herein, is within the scope of the invention.
In another embodiment of the different aspects of the invention, an endogenous plant Hsf may be overexpressed according to the methods and uses of the invention. For example, a tomato Hsf may be overexpressed in tomato, a wheat Hsf may be expressed in wheat, a rice Hsf may be overexpressed in rice. Plants and their one or more Hsf may be selected from any plant, such as from one of the families or species listed above.
Overexpression according to the invention means that the transgene is expressed at a level that is higher than expression driven by its endogenous promoter. For example, overexpression may be carried out using a strong promoter, such as the cauliflower mosaic virus promoter (CaMV35S), the rice actin promoter or the maize ubiquitin promoter or any promoter that gives enhanced expression. Alternatively, enhanced or increased expression can be achieved by using transcription or translation enhancers or activators and may incorporate enhancers into the gene to further increase expression. Furthermore, an inducible expression system may be used, such as a steroid or ethanol inducible expression system. The coding sequence may be on a monocistronic or polycistronic messenger RNA. Also envisaged is ectopic expression, i.e. gene expression in a tissue in which it is normally not expressed
According to the different aspects of the invention, plant characteristics are increased or improved. This is understood to mean an increase or improvement in plant productivity, water use efficiency, water productivity, drought tolerance or pathogen resistance compared to the level as found in a wild type plant.
According to one embodiment of the first aspect of the invention, the method increases water productivity. Thus, the method can be used to increase water productivity.
As used herein, water productivity describes the amount of yield produced per unit of water (for example ml or l) used. The transgenic plants as described herein require a lower amount of water than a wild type plant to produce the same amount of yield under normal non drought conditions where water is not at a shortage. Thus, according to the invention, water productivity can be improved under non drought conditions. For example, water productivity can be improved under non drought conditions by expression of the Arabidopsis Hsf is AtHSFA1b in another plant as defined herein.
In a different embodiment of first aspect of the invention, the method improves plant productivity under water deficit conditions. Thus, the method of the first aspect confers plant drought tolerance.
Water deficit or water limited conditions as used herein refer to conditions where water is at a shortage. This includes conditions where water is at a shortage compared to the normal average of water available to a plant grown in the particular environment, for example due to a change in climate or unseasonable weather. It also refers to conditions where water is generally known to be scarce, for example in arid climatic zones. Water shortage for a prolonged period of time is known as drought.
In another embodiment of the method of the first aspect of the invention, the method of the invention confers pathogen resistance. Plants with ability to resist infection by a particular pathogen are referred to as having increased resistance to that pathogen. Pathogens according to the different aspects of the invention include any viral, bacterial, fungi or animal pathogens, such as nematodes or insects, which infect plants. In one embodiment, the pathogen may be Pseudomonas syringae pv. Tomato, turnip crinkle virus or Hyaloperonospora parasitica. Fungal pathogens according to the invention include, but are not limited to the rust fungi (order Uridenales) e.g. Puccinia graminis, Puccinia striiformis (yellow rust) P. recondite and other Puccinia species, flax rust (Melampsora lini); Rhizoctonia sp. or Phakospora pachyrhizi (Soybean rust), the powdery mildew fungi (order Erysiphales, e.g barley powdery mildew (Blumeria graminis); Erisyphe sp. (infects legumes, trees and shrubs), Leveillula sp. (infects Solanaceae), Golovinomyces sp. (infect Cucurbits and Compositae), Podosphaera sp. (infects Rosaceae); Fusarium sp. Verticillium sp., Rice blast fungus Magnoporthe grisea or Potato blight (Phytophtora infestans).
Bacterial pathogens according to the invention include, but are not limited to Pseudomonas syringae (various pathovars), Xanthomonas sp. (e.g. X. campestris infects Brassicas, X. axonopodis causes citrus canker).
Viral pathogens according to the invention include, but are not limited to Tobacco mosaic virus (Solanaceae), tomato spotted wilt virus, rice tungrovirus, maize rough dwarf virus. Maize streak virus, cucumber mosaic virus, potato viruses X and Y, brome mosaic virus, pepper mild mottle virus, pea seed borne mosaic virus or pea ennation virus.
In a second aspect, the invention relates to a method for improving water use efficiency in plants comprising introducing and over-expressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant. In particular, water use efficiency can be improved under non drought conditions. For example, water use efficiency can be improved under non drought conditions by expression of the Arabidopsis Hsf is AtHSFA1 b in another plant as defined herein.
The term water use efficiency as used herein relates to the plants ability of using a water supply efficiently under normal or water deficit conditions. Because the plants according to the invention use water more efficiently than a wild type plant, they show drought resistance and thus prolonged lifespan under water limiting conditions. However, the inventors have also surprisingly found that the plants according to the invention use water more efficiently under normal non-drought conditions compared to wild type plants. As shown in the examples, plants according to the invention require less amount of water than wild type plants to survive and produce yield, thus they use the water supply more efficiently. It will be appreciated that the term normal conditions refers to conditions which are not exceptional, i.e. conditions in which water is not limited. Drought conditions are not normal conditions as water is at a deficit. It will also be appreciated that what in detail is to be understood by normal conditions depends on the plant concerned and on the climatic zone in which the plant is grown.
Thus, in one embodiment of the second aspect of the invention, the method increases water productivity. The method as described in the second aspect of the invention can thus be used to increase water productivity.
In another embodiment, the method improves water use efficiency under water deficit conditions. Therefore, the method increases plant drought tolerance. As shown in examples 2 to 7, plants transformed with a gene sequence encoding a plant HSF polypeptide whose expression is regulated by a strong promoter have improved resistance to prolonged periods of water shortage, i.e. drought conditions. Wild type plant survival rates are very low under these conditions whereas the transgenic plants survive and produce yield.
In another aspect, the invention relates to a method for increasing water productivity. Water productivity can be increased under normal conditions, i.e. conditions where water is not limited. In a further aspect, the invention provides a method for conferring drought resistance
In a further aspect of the invention, the invention relates to the use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving plant productivity.
Another aspect of the invention relates to the use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving water use efficiency. In particular, this use relates to improving water use efficiency under normal non drought conditions or under drought conditions. Therefore, according to the invention, a plant Hsf can be used to improve water productivity, thereby enabling the plant to use less water than a wild type plant. Thus, the amount of water used in irrigation of crop plants can be reduced. In addition, the use according to the invention also provides that a plant Hsf can be used to improve water use efficiency under water deficit conditions, such as drought conditions.
The invention also provides the use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving water productivity and the use in conferring drought tolerance.
In a final aspect, the invention provides the use of a polynucleotide sequence comprising or consisting of a plant Hsf in conferring pathogen resistance. The pathogen may be selected from those described herein.
The invention is further described by reference to the non-limiting figures and examples.



FIGURES
FIG. 1 shows SEQ ID No 1, the full length genomic sequence of AtHSFA1b.
FIG. 2 shows the conserved domains of Arabidopsis Hsfs.
FIG. 3 shows wild type and 35S-Hsf3 (left) plants which were not watered for 2 weeks. Transgenic plants are marked with an arrow.
FIG. 4 shows plants which were not watered for 2.5 weeks and then rewatered. The picture was taken 48 hours after rewatering. Transgenic plants are marked with an arrow.
FIG. 5a shows a decline in pot water content for 35S-Hsf3 (HSF3) and wild type (WT) plants under drought conditions in a glasshouse. Withdrawal of water began at day 0. Re-watering commenced when the pot water content had attained 33 g (indicated by the arrows). Note that this took 24 hours longer for 35S-Hsf3 plants.
FIG. 5b shows the quantum efficiency of photosynthetic electron transport (Fv/Fm) in 35S-Hsf3 versus wild type plants in watered conditions (WT-w and HSF w) and just prior to recommencing watering as indicated (WT d and HSF d).
FIG. 6 shows the total seed yield after 10-11 days of drought and then subsequent re-watering until seed set. Under well-watered conditions, in both sets of conditions, 35S-Hsf3 plants yield better than WT. This differential is maintained after a moderate drought stress.
FIG. 7 shows the vegetative and reproductive biomass as well as seed yield of plants kept at different soil water contents (40% and 80%). In both soil water contents Hsf3 plants show higher seed yield and reproductive biomass, but reduced vegetative biomass.
FIG. 8ashows wild type (col-0) and transgenic plants overexpressing Hsf3 infected with virulent Pseudomonas syringae pv. tomato DC3000. Diseased leaves were scored as those showing a yellowing 7 days after inoculation. FIG. 8b shows the number of bacteria (colony forming units) recovered after 5 days of infection. *=significant difference (p<0.05).
FIGS. 9a and 9b are bar graphs showing wild type and transgenic plants overexpressing Hsf3 infected with Hyaloperonospora parasitica. Disease symptoms were scored by different disease classes as follows: I healthy leaves, II chlorotic lesions, III leaves with sporulation and IV leaves with chlorotic lesions and sporulation. The HSF3 plants have mainly healthy leaves and at most leaves that show chlorotic lesions. The fungus is prevented from sporulating on HSF3 leaves. As shown in FIGS. 9c and 9d, callose deposition at pathogen entry point was scored using epifluorescence microscopy by counting the coincidence of spore presence and callose deposition. The more callose with spores the higher the resistance, as seen in the 35S-Hsf3 plants.
FIG. 10 Northern blot analysis. Wild type and transgenic plants overexpressing Hsf3 infected with turnip crinkle virus. Viral RNA accumulation in leaves of HSf3 plants is delayed compared with wild type.
FIG. 11 shows foliar H2O2 levels during rosette development in 358-Hsf3 (HSF) and wild type plants (WT).
FIG. 12 shows a decline in pot water content for 35S-Hsf3 (HSF3) and wild type plants (C24) under drought conditions in a glasshouse. Withdrawal of water began at day 0.
FIGS. 13a and 13b show a comparison of wild type and transgenic plants overexpressing Hsf3 infected with virulent Pseudomonas syringae pv. tomato DC3000. FIG. 13a shows diseased leaves scored as those showing a yellowing 7 days after inoculation. FIG. 13b shows number of bacteria (cfu/cm2) recovered after 5 days of infection.
FIGS. 14a to 14c are bar graphs showing wild type and transgenic plants overexpressing Hsf3 infected with Hyaloperonospora parasitica. Disease symptoms were scored by different disease classes as follows: I healthy leaves, II chlorotic lesions, III leaves with sporulation and IV leaves with chlorotic lesions and sporulation. The fungus is prevented from sporulating on HSF3 leaves as shown in FIG. 14b. FIG. 14c shows callose deposition at pathogen entry point scored using epifluoresnce microscopy by counting coincidence of spore presence and callose deposition.
FIG. 15 compares wild type and transgenic plants overexpressing Hsf3 infected with virulent Pseudomonas svringae pv. tomato DC3000 DC3000. Diseased leaves were scored as those showing a yellowing 7 days after inoculation. * = significant difference (p<0.05).
FIG. 16 shows foliar H202 levels during rosette development in 358-Hsf3 and wild type plants.
FIG. 17 is the expression construct used to transform Brassica napus. 
FIG. 18 shows a measurement of H2O2 of primary Brassica napus transgenics transformed with the construct as shown in FIG. 17
FIG. 19 shows that PR1 gene expression is significantly higher in the 35S:HSF3 12.2 line compared with controls. Gene expression data was analysed in the primary Brassica napus transgenics transformed with the construct as shown in FIG. 17 using qRT-PCR.
FIG. 20 shows APX2 gene expression in Brassica napus transgenics transformed with the construct as shown in FIG. 17.



EXAMPLES
35S-Hsf3 (35SAthsfA1b) plants display a number of characteristics which can be classified into two major traits, both elicited by overexpression of AtHsf3 and which interact to improve water productivity of these plants.
The plants show altered growth and development under well-watered conditions that mimic plants growing under mild water deficit conditions. Under a range of water deficit conditions these characteristics ensure that Hsf3 plants out-perform wild type plants in terms of seed yield and survival. Plants show increased biomass of reproductive structures and seed production at the expense of leaf biomass, under both well-watered and drought conditions. The early flowering of Hsf3 plants under both well-watered and drought stress, altered senescence of source leaves and the consequent maintenance of photosynthetic efficiency are key factors in the increased seed yield of 35S-AtHSF3 plants under both well-watered conditions and a range of soil water deficit conditions.
35S-AtHsf3 (35SAthsfA1b) plants also show reduced transpiration. An elevated apoplastic H2O2 level in plants over-expressing Hsf3 may, in part, be responsible for the trait by altering stomatal guard cell function such that water loss through stomatal pores is reduced. H2O2 produced in guard cells has been implicated in the ABA signal transduction pathway leading to stomatal closure. Without wishing to be bound by theory, the inventors suggest that apoplastic H2O2 from elsewhere in the leaf can also influence guard cell function. Importantly, in these plants reduced rates of transpiration did not lead to a sufficiently reduced CO2 assimilation rate as to reduce growth and yield.
Example 1
Creating a Construct and Transforming Plants
The creation of the plant described here has been published (Prandl et al 1998). Briefly, a full length 1.7 kb cDNA fragment containing the entire Hsf3 coding sequence was inserted as a BamH1 fragment into the binary Ti vector pB1121, base on the well-known vector pBIN19. This procedure fused the cDNA to the CaMV 35S promoter. The mRNA would also contain a GUS coding sequence followed by a nos polyadenylation sequence. Moncistronic constructs fused to 35S promoter have also been created for transformation into crop plant species.
Example 2
Plant Growth in Response to Drought and Well Watered Conditions
Plants were grown in controlled environment rooms under short day (8 h light/16 h dark) at 22° C. and 60% relative humidity. The Wild type (right) and 35S:AtHSF3 (left) plants are shown in FIG. 3. The plants were not watered for 2 weeks in a controlled environment room. These plants were typical in their response.
As shown in FIG. 4, the 35S AtHSF3 plants recover sufficiently from prolonged drought stress so that upon re-watering they flower and set seed. Wild type plants do not recover.
It was also shown that 35S-AtHSF3 plants have higher leaf temperatures. Under well-watered conditions, 35S-AtHSF3 plants have a 1.5-2.5° C. higher leaf temperature, implying a reduced transpiration rate and a lower stomatal conductance.
Plants were grown in individual pots for 4 weeks in the controlled environment room as described above. After 4 weeks plants were transferred to the glass house under long day and water was withdrawn for half of the pots. The plants were left to dry out to a similar soil water content of  ̃35% and were then re-watered.
When control and 35S-AtHsf3 plants were re-watered at the times indicated in FIG. 5, seed yield was substantially higher in 35S-AtHsf3 plants than wild type (WT) plants (Table 3), a differential that was also observed in watered controls (Table 3). Since the amount of water provided was the same, these data represent a real increase in water productivity of the 35S-AtHsf3 plants compared with WT control. Thus, 35S-AtHsf3 plants have a higher seed yield in well-watered conditions and after a mild water deficit stress.







TABLE 3







Total seed yield from plants subjected


to drought in a containment glasshouse.













Seed Yield




Genotype
Treatment
(mg plant1)
n
P














35S-AtHSF3
Watered
361 ± 84
4
0.022


WT
Watered
156 ± 86
4


35S-AtHSF3
mild water deficit
368 ± 31
4
0.0005


WT
mild water deficit
156 ± 38
4





The water stress conditions were those applied in FIG. 5a up to the times indicated, when re-watering started. Controls were kept well-watered throughout the experiment.






Furthermore, rosette and reproductive structure biomass was measured. This was done by at the end of the life cycle. Plants were bagged and these bags contained all stem, pods, seeds etc above the rosette. The rosettes were harvested and bagged separately. The seed was threshed from the stems. All chaff was collected. The chaff, rosettes and soil pots were placed in a drying oven at 70 degrees Celsius for 72 hrs. These were weighed. Seed was weighed and counted. Dry weight measurements of the above ground vegetative (rosette) and reproductive (stalks and pods) parts and seed yield were determined under two different soil water contents. (see FIG. 7)
The experiments shows that 35S-AtHsf3 rosette biomass was reduced but the biomass of all aerial parts was increased in the same plants.
Example 3
Measurement of Photosynthetic Electron Transport
This was done by measuring the maximum efficiency (Fv/Fm) of photosystem II. Whole rosette or leaf Fv/Fm values were taken to indicate their response to the drought treatment using a chlorophyll a fluorescence imaging system (Fluorimager; Technologica Ltd, Colchester, UK) as described by Barbagallo et al 2003.
CO2 gas exchange measurements were carried out on leaves of 5 week old plants using a CIRAS-2 (PP systems, Hitchin, U.K) CO2/H2O Infra Red Gas Analyser. Photosynthetic electron transport rates higher in drought-stressed 35S-AtHSF3 plants. The quantum efficiency of photosynthetic electron transport showed no difference between the genotypes in watered conditions, but was 15% better after 11 days of drought in 35S-HSF3 versus wild type (see FIG. 5b). There were no differences between 35S-AtHSF3 and wild type in the response of photosynthetic CO2 exchange to intercellular CO2 concentration when well watered. These parameters were used to evaluate transgenic plants.
Example 4
Measurement of Flowering
Growth conditions were as described earlier. The first 4 weeks plants were grown in controlled environment rooms after which plants were transferred to the glass house and water level was maintained at either 40% or 80% soil water content. Measurement of flowering time was done by noting the time of the visible (by naked eye) of first appearance of the floral apical meristem as the number of days post germination until the terminal flower opened. 35S-Hsf3 plants fully flowered (terminal flower open) on average 7 days earlier than the Col-0 plants.
It was shown that 35S-AtHsf3 plants showed on average 7 days early flowering. During flowering and seed set, leaves of 35S-AtHSF3 plants show delayed senescence until siliques yellowed under glasshouse conditions. This may mean that in 35S-Hsf3 flowering plants, source leaves could maintain a supply of photosynthate to ensure an increased reproductive biomass and the supply to developing seed for longer, and this was a contributing factor for the observed increase in seed yield in 35S-AtHsf3 plants, and reproductive biomass.
Example 5
Measurement of H2O2 Content
100 mg of leaf material was extracted in 0.1M HCl and the supernatant of this extraction was purified using activated charcoal. Analysis of H2O2 levels was by spectrophotometry using the Amplex Red kit from Invitrogen. It was shown that the 35S-AtHsf3 plants have up to 3 times the foliar H2O2 content of wild type plants. This increased H2O2 accumulates only in the apoplast and is generated by enhanced activity of apoplastic reticuline (carbohydrate) oxidase. The enhanced H2O2 levels stimulate an increase in both ascorbate peroxidase and cell wall peroxidase activities.
Example 6
Measurement of ABA Content
1 g of leaf material was harvested and extracted in methanol. The supernatant of this extraction is dried down and samples were dissolved in diethylether/methanol. The diethylether phase is put through a NH2 SPE column and washed subsequently with chloroform/isopropanol before resuspending in diethylether and acetic acid. Analysis of ABA content was carried out using Gas chromatography combined with mass spectrometry (GC/MS) adapted from a method described by (Muller et al 2002).
The experiments showed that ABA content is no different from wild type plants under well-watered conditions and significantly our microarray data show no alteration of ABA-responsive genes (other than APX2).
Example 7
Microarray Experiment
Rosettes from 5 week old Col-0 and HSF3 plants were harvested and RNA was extracted from 2 different biological samples. Gene expression analysis was carried out using Arabidopsis 3 whole genome oligonucleotide-based microarrays from Agilent cRNA labelling and hybridisation of the arrays was according to the manufacturers' instructions.












TABLE 4










Hsf3/WT



Name
Locus
Classification
fold change






















At5g03720
At-HSFA3
3.3



HSF4
At4g36990
At-HSFB1
2.5



HSF5
At1g67970
At-HSFA8
2.1



HSF6
At5g62020
At-HSFB2a
2










Example 8
Pathogen Infections
Pseudomonas syringae pv tomato (DC3000-) infection was carried out on 5 week old plants. Whole rosettes were dipped in a solution containing 5*107 colony forming units (cfu)/ml. Leaf material was harvested at the beginning (day 0) and 5 days post inoculation to determine the bacterial proliferation by grinding in MgSO4 and plating a dilution series on KB plates containing Rifampicin (50 mg/L) and Cycloheximide (100 mg/L). Bacterial proliferation is calculated as the difference in cfu between day 5 and day 0. Symptoms were scored 6 days post inoculation (yellowing leaves).
Hyaloperonospora parasitica infection was carried out at a concentration of 5*104 spore/ml. The fungal spores are obtained from leaves of infected plants and are extracted in water and diluted to the right concentration. Leaves of 3 week old plants are inoculated with the fungus by spraying a fine layer of liquid onto each leaf. Disease symptoms and callose formation are scored 7 days post inoculation.
Turnip crinkle virus infection was carried out on 3 week old plants. 2.5 ul of 0.1 ug/ul viral RNA in bentonite buffer were gently rubbed into three leaves of each plants. Systemic leaves are harvested at different times post inoculation and RNA is extracted and checked for viral RNA replication via Northern blotting. As a loading control, the blot is probed with 18S rRNA.
Example 9
Rapeseed (Brassica napus), also known as rape or oilseed rape, is a bright yellow flowering member of the family Brassicaceae (mustard or cabbage family). Rapeseed is grown for the production of animal feed, vegetable oil for human consumption, and biodiesel; leading producers include the European Union, Canada, the United States, Australia, China and India. In one set of experiments, a construct overexpressing HSF3 from Arabidopsis was used to transform rapeseed.
Brassica napus ecotype Q6 was transformed with a 35S:A.t.HSF3 construct and an empty vector control (35S:Kan) (see FIG. 17). Eight months post transformation, 7 empty vector plants and 2 HSF3 transformed lines were recovered onto soil. Transformants were screened for the presence of the transgene using a 35S forward and HSF3 reverse primer, using 35S: Kan, empty vector controls and Arabidopsis HSF3 plant DNA as a positive control. Only one of the HSF3 transgenics (35S:HSF3 12.2) amplifies the 35S-HSF3 junction PCR product. The empty vector controls are also negative.
H2O2 of primary transgenics was measured (see FIG. 18). Foliar H2O2 levels are increased in line 35S:HSF3 12.2 compared to the empty vector controls.
Stomatal conductance and photosynthetic rate of the primary transgenics were also measured. Stomatal conductance is reduced in the 35S:HSF3 12.2 plant. Despite their reduction in stomatal conductance, the linear phase of photosynthesis is not affected in the 35S:HSF3 12.2 plants, however, photosynthesis saturates at lower levels compared to the empty vector controls and the second HSF3 line.
We also analysed the transgenic plants by thermal imaging of primary transgenic lines of oils seed rape. It was found that the HSF3 transgenic (35S:HSF3 12.2) shows a 1° C. warmer leaf temperature across the plant compared with flanking empty vector controls. This indicates less evaporative water loss by transpiration in the HSf3 transgenic compared with controls. This agrees with the lowered stomatal conductance values for the same line and is consistent with the observations made in 35S:HSF3 Arabidopsis plants.
Furthermore, gene expression data was analysed in the primary transgenics using qRT-PCR. PR1 gene expression is significantly higher in the 35S:HSF3 12.2 line compared with controls. This indicates activated pathogen defences.
We also analaysed APX2 gene expression. Increased expression in the 35S:HSF3 12.2 line shows functioning HSF3 and, from a physiological point of view, again indicates a change in leaf water status.
The data demonstrates that transformation of Brassica napus with a 35S:A.t.HSF3 construct overexpressing HSF3 from Arabidopsis produces similar results to those observed when overexpressing HSF3 from Arabidopsis in Arabidopsis. 
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## Claims

1. A method of generating a plant having improved water productivity under non-drought and water deficit conditions, the method comprising
(a) introducing to the plant and overexpressing in the plant a polynucleotide comprising a sequence encoding a Class A plant heat shock factor (Hsf), thereby producing a transgenic plant;
(b) exposing the transgenic plant to non-drought conditions or water deficit conditions; and
(c) assessing the yield produced from the transgenic plant per unit of water consumed, wherein a greater yield, relative to that of a corresponding wild type plant, indicates the generation of a plant having improved water productivity under non-drought and water deficit conditions.

2. The method of claim 1, wherein the non-drought conditions are mild water deficits.

3. The method of claim 1, wherein the water deficit conditions are drought conditions.

4. The method of claim 1, wherein the plant is a cereal plant.

5. The method of claim 1, wherein the plant is maize, wheat, rice, oilseed rape, sorghum, soybean, cotton, potato, tomato or poplar.

6. The method of claim 1, wherein the Class A plant Hsf is an Arabidopsis plant Class A Hsf.

7. The method of claim 6, wherein the Arabidopsis plant Class A Hsf is AtHsfA1a, AtHsfA1b, AtHsfA1d, AthsfA1e, AtHsfA2, AtHsfA3, AtHsfA4a, AtHafA4c, AtHsfA5, AtHsfA6a, AtHsfA6b, AtHsfA7a, AtHsfA7b, AtHsfA8 or AtHsfA9.

8. The method of claim 7, wherein the Arabidopsis plant Hsf is AtHsfA1b or AtHsfA1a.

9. The method of claim 1, wherein the Class A plant Hsf is a tomato plant Class A Hsf.

10. The method of claim 6, wherein the Class A plant Hsf is the Arabidopsis plant Hsf AtHsfA1b, which is represented by SEQ ID NO:1.

11. The method of claim 6, wherein the Arabidopsis plant Hsf is AtHsfA1b, which is represented by SEQ ID NO:1.