Plant sugar sensors and uses thereof

Details Plant sugar sensors and uses thereof Plant sugar sensors and uses thereof

1996-03-25

2003-10-14

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C435S410000, C435S419000, C435S320100, C435S455000, C536S023100, C536S023200, C536S023600, C800S281000

Reexamination Certificate

active

06632602

ABSTRACT:

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made in part with Government funding, and the Government therefore has certain rights in the invention.
BACKGROUND OF THE INVENTION
This application relates to plant carbohydrate metabolism; in particular, to enzymes which transduce sugar-sensing signals, their encoding genes, and uses thereof.
Sugars are said to be regulatory molecules that are capable of controlling physiology, metabolism, cell cycle, development, and gene expression. Throughout the higher plant life cycle, from germination to flowering to senescence, sugars affect growth and development. Recently, it has become apparent that sugars are physiological signals capable of repressing or activating plant genes that are involved in many essential processes, including photosynthesis, the glyoxylate cycle, respiration, starch and sucrose synthesis and degradation, nitrogen metabolism and storage, pathogen defense, the wounding response, cell cycle progression, pigmentation, and senescence (Sheen,
Photosynthesis Res
. 39, 427 (1994); Thomas and Rodriguez,
Plant Physiol
. 106, 1235 (1994); Knight and Gray,
Mol. Gen. Genet
. 242, 586 (1994); Lam et al.,
Plant Physiol
. 106, 1347 (1994); Chen et al.,
Plant J
. 6, 625 (1994); Reynolds and Smith,
Plant Mol. Biol
. 29, 885 (1995); Herbers et al., Plant Mol. Biol. 29, 1027 (1995); Mita et al.,
Plant Physiol
. 107, 895 (1995)). Studies in a variety of plant species have also shown that sugar homeostasis appears to be tightly regulated. Elevated sugar concentration leads to stunted growth, reduced photosynthesis, leaf curling, chlorosis, necrotic leaves, and anthocyanin accumulation (Casper et al.,
Plant Physiol
. 79, 11 (1985); von Schaewen et al.,
EMBO J
. 9, 3033 (1990); Dickinson et al.,
Plant Physiol
. 95, 420 (1991); Tsukaya et al., Plant Physiol. 97, 1414 (1991); Sonnewald et al.,
Plant J
. 1, 95 (1991); Huberet and Hanson,
Plant Physiol
. 99, 1449 (1992); Sonnewald et al., Plant Responses to Sugar Accumulation in Transgenic Tobacco Plants, pp. 246-257, In: M. A. Madore, W. J. Lucas (eds.),
Carbon Partitioning and Source
-
Sink Interactions in Plants
, American Society of Plant Physiologists, Rockville, Md., (1995)). In addition, environmental factors such as elevated CO
2
and intrinsic genetic variations such as different invertase levels have been proposed to affect photosynthetic capacity through sugar regulation (Stitt,
Plant Cell Environ
. 14, 741 (1991); Stitt et al.,
Planta
183, 40 (1991); VanOosten et al.,
Plant Cell Environ
. 17, 913 (1994); Nie et al.,
Plant Physiol
. 108, 975 (1995); Goldschmidt and Huber,
Plant Physiol
. 99, 1443 (1992)).
SUMMARY OF THE INVENTION
By manipulating the expression of a plant hexokinase protein (HXK), we have discovered that this protein is a sensor that mediates diverse sugar responses in plants. In particular, we have engineered transgenic plants that either: (a) express a decreased level of hexokinase protein due to expression of an antisense hexokinase gene and therefore exhibit a decreased sensitivity to sugar; or (b) express an increased level of hexokinase protein and therefore exhibit an increased sensitivity to sugar. Our discovery has broad implications for the manipulation of agricultural crops, for increasing crop yield and quality, and for reducing production costs.
In general, the invention features a method for reducing the level of a plant hexokinase protein in a transgenic plant cell, the method involving expressing in the transgenic plant cell (for example, a cell from a monocot, a dicot, or a gymnosperm) an antisense hexokinase nucleic acid sequence. This produces transgenic plants that are less sensitive to sugar (for example, glucose, sucrose, fructose, or mannose).
In preferred embodiments, the antisense hexokinase nucleic acid sequence is encoded by a transgene integrated into the genome of the transgenic plant cell; the antisense hexokinase nucleic acid sequence includes a plant antisense hexokinase DNA sequence (for example, a sequence that is based on the AtHXK1 nucleotide sequence of
FIG. 1F
(SEQ ID NO: 3) or the AtHXK2 nucleotide sequence of
FIG. 1G
(SEQ ID NO: 4)); and the method further includes growing a transgenic plant from the transgenic plant cell, whereby the level of the hexokinase protein is reduced in the transgenic plant.
In related aspects, the invention features a plant cell (for example, a plant cell from a monocot, dicot, or gymnosperm) expressing an antisense hexokinase nucleic acid sequence; and a plant expression vector including an antisense hexokinase nucleic acid sequence, wherein the sequence is operably linked to an expression control region.
In yet another aspect, the invention features a method for increasing the level of a hexokinase protein in a transgenic plant cell, involving expressing in the transgenic plant cell a hexokinase nucleic acid sequence. In preferred embodiments, the hexokinase nucleic acid sequence is from a plant (for example, a DNA sequence that is identical to the AtHXK1 nucleotide sequence of
FIG. 1F
(SEQ ID NO: 3) or that is substantially identical to the AtHXK2 nucleic acid sequence of
FIG. 1G
(SEQ ID NO: 4)). This method produces transgenic plants having an increased sensitivity to sugar.
In related aspects, the invention features a substantially pure plant HXK polypeptide including an amino acid sequence substantially identical to the amino acid sequence of AtHXK1 (SEQ ID NO: 1) or AtHXK2 (SEQ ID NO: 2). In preferred embodiments of both of these aspects, the HXK polypeptide is obtained from a plant including, but not limited to, a monocot (for example, rice, corn, wheat, or barley), a dicot (for example, a member of the Solanaceae (for example, potatoes) or a member of the Cruciferae (for example, Arabidopsis, broccoli, cabbage, brussel sprouts, rapeseed, kale, Chinese kale, cauliflower, or horseradish)), and a gymnosperm.
In yet other related aspects, the invention features a substantially pure DNA encoding a plant HXK polypeptide that includes an amino acid sequence substantially identical to the amino acid sequence of AtHXK1 (SEQ ID NO: 1) or AtHXK2 (SEQ ID NO: 2). In preferred embodiments, the DNA includes the nucleotide sequence shown in
FIG. 1F
(SEQ ID NO: 3) or includes a nucleotide sequence that is substantially identical to the sequence that is shown in
FIG. 1G
(SEQ ID NO: 4). Such DNAs are obtained from any plant including, but not limited to, a monocot (for example, rice, corn, wheat, and barley), a dicot (for example, a member of the Solanaceae (for example, potatoes) or a member of the Cruciferae (for example, Arabidopsis, broccoli, cabbage, brussel sprouts, rapeseed, kale, Chinese kale, cauliflower, or horseradish)), and a gymnosperm. In other preferred embodiments, the DNAs of the invention are operably linked to a constitutive or regulated promoter.
In yet other related aspects, the invention features a vector including any of the substantially pure DNAs of the invention, the vector being capable of directing expression of the protein encoded by the DNA in a vector-containing cell; a cell, for example, a prokaryotic cell (for example, an
E. coli
cell) or a eukaryotic cell (for example, a plant cell) which includes any of the DNAs of the invention; and a transgenic plant (or a cell or a seed derived from such a transgenic plant) including any of the DNAs of the invention integrated into the genome of the plant, wherein the DNA is expressed in the transgenic plant.
In various preferred embodiments, the plant cell contains the DNA in the sense orientation and has an increased sensitivity to sugar; the plant cell contains the DNA in the antisense orientation and is less sensitive to sugar; and the DNA is expressed under the control of a constitutive promoter or regulated promoters.
In two other aspects, the invention features a method of producing a plant HXK polypeptide involving: (a) providing a cell transformed with a gene encoding a polypeptide including either an amino acid sequence substantially identical to the amino acid sequence of

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