(WO/2001/017333) TRANSGENIC FIBER PRODUCING PLANTS WITH INCREASED EXPRESSION OF SUCROSE PHOSPHATE SYNTHASE
- Biblio. Data
- Description
- Claims
- National Phase
- Notices
- Documents
- Note: OCR Text
- Note: Text based on automatic Optical
Character Recognition processes. Please
use the PDF version for legal matters
- Note: Text based on automatic Optical
TRANSGENIC FIBER PRODUCING PLANTS WITH INCREASED EXPRESSION OF SUCROSE PHOSPHATE SYNTHASE FIELD OF THE INVENTION The present invention relates to a method for increasing the yield or quality of product from a plant by altering the expression of sucrose phosphate synthase. In particular, the present invention provides a transgenic cotton plant that has an increased level of sucrose phosphate synthetase relative to a non-transgenic cotton plant. Methods are also provided for increasing the yield or the quality of cotton fiber and the yield of cotton seed produced from a cotton plant. General methods are provided for regulating the thickness of cell walls, for increasing the yield and quality of other plant fibers, for regulating the ratio of cellulose to other dry weight components of the plant, for increasing seed yield, and for increasing tolerance of photosynthetic efficiency to cool night temperatures.
BACKGROUND OF THE INVENTION
The control of high-rate cellulose production and its regulation by temperature are
critical to agriculture, since all plant growth (and hence the production of all food crops)
depends on cellulose synthesis to build cell walls throughout the vegetative and
reproductive parts of the plant. The cellulose within the primary walls of all cells of the
plant body is also of direct industrial importance as a digestible part of animal forage and
for manufacture of thickeners, ethanol, and other cellulose-based or cellulose-derived
products. Furthermore, plant parts based on secondary cell walls with high cellulose
content are contained in or compose economically important plant products, including
cotton fibers, wood, and fibers in forage crops. The agronomic productivity and product
quality of wood and cotton, as well as other fiber crops such as hemp and flax, are in
large part determined by the biosynthesis of cellulose. Therefore, an understanding of the
basic regulatory
Since cotton fiber weight is more than 90% cellulose, cotton is one particular crop
where enhancing the flow of carbon to cellulose production can increase yield and
Cotton fiber yield is the most important determinant of the value of the crop to the
producer. Reputable cotton breeders have recently pointed out that cotton production has
reached a fiber yield plateau, which bodes ill for the financial success of producers given
escalating costs. Potential contributors to this problem include the environmental
sensitivity of cotton fiber and seed development, the narrow genetic base of commercial
cotton, and the recent introduction of transgenic traits such as herbicide and insect
resistance through back-crossing with transformed
Coker
Similarly, seed yield is of value to the cotton producer since seeds are sold for oil production and animal feed. Another minor component, the short fuzz fibers on each seed, provides added economic value to the seed crop. Increased seed and fuzz fiber yield without sacrifice of lint fiber yield or quality would help the producer recover more profit per acre of cotton production. As for cotton seed, increased yield of any seed crop will be of major benefit to agriculture.
Improved cotton fiber quality parameters such as micronaire, maturity ratio,
length, length uniformity, bundle strength, and single fiber strength are desired by the
textile industry to produce increasingly high quality products and to take full advantage of
modern spinning technologies. Fiber quality parameters should also be high enough for
Other plant fibers, although often of different tissue origin, share structural features in common with cotton fibers in being elongated cells with cellulose-rich walls.
Like cotton fibers, other plant fibers of industrial use are required to have high quality as defined by factors such as cellulose content and wall thickness, diameter, fineness (or coarseness), length, strength, durability, uniformity, elasticity, and elongation. There is an optimum range of such parameters for each particular fiber source and industrial use.
Taking examples from wood fibers used after pulping in paper production, longer fiber length and higher single fiber elongation both promote higher paper tear strength. In addition, thick fiber walls promote high pulp yield and production of absorbent paper with high tearing resistance. However, thinner fiber walls promote fiber collapse and better inter-fiber bonding that aids production of high quality writing paper. Therefore, there exists a need to control cell wall thickness and other fiber quality parameters in either negative or positive directions in diverse fibers to improve their yield or quality or expand the range of their industrial utility.
Maximizing crop productivity and utility per acre is a key component of sustainable agriculture. Enhanced production of multiple products from the same crop, such as seed and fiber, would be useful. Similarly, it will be an advantage to maximize the possibility of a successful crop harvest, for example by generating plants with stiffer stems that can better resist lodging in the field without sacrificing the yield of a seed crop.
An increasing level
Cotton leaves assimilate most carbon into starch during the day, and the starch is
converted to sucrose at night for translocation to sinks. As just described, cotton fibers
are not well adapted to use this sucrose efficiently for cellulose synthesis during cool
nights. Therefore, cool nights reduce cotton photosynthetic efficiency during the
following warm day (Warner et al.,"Response of Carbon Metabolism to Night
Temperatures in
Sucrose phosphate synthase ("SPS") is a key protein involved in carbon
metabolism in plants (See Figure 1). SPS catalyzes the formation of sucrose phosphate
from UDP-glucose and fructose 6-phosphate. In the leaf, SPS is important in controlling
the partitioning of reduced carbon between starch and translocatable sucrose (Huber et al.,
"Role and Regulation of Sucrose-Phosphate Synthase in Higher Plants,"Annu. Rev. Plant
Physiol. Plant Mol.
Its level of activity can regulate the amount of metabolic flux directed toward cellulose synthesis compared to respiration (See Figure 2). According to this model, SPS within cellulose-storing sink cells can increase sink strength through an enhanced rate of cellulose synthesis by promoting sucrose synthesis in one or both of two cases: (a) if sucrose transported from the leaves is cleaved to release glucose and fructose before or after entering the sink cells ; and/or (b) to reuse the fructose released by the activity of sucrose synthase to channel UDP-glucose and fructose to cellulose synthase. A decreased level of SPS activity can decrease sink strength, by analogous mechanisms, in any case where sink filling is affected by sucrose levels.
In tomato, over-expression of SPS has been shown sometimes to cause
It should be noted that although cotton bolls and tomatoes are both classified botanically as fruits, the nature of the fruits and the relative importance of the seeds they contain is very different. Tomato fruits are essentially sacks of primary cell walls filled with water and soluble glucose, fructose, and sucrose as storage carbohydrates. These sugars crystallize upon drying, contributing to fruit dry weight. Within the fruit, tomato seeds are not a significant sink due to their small size, and they have no economic value except for propagation of tomato. The fruit is the major sink in tomatoes; it constitutes almost all of tomato yield and is the only tomato part with significant economic value.
In contrast, the cotton fruit is relatively dry and thin-walled. The fruit itself does not constitute any substantial sink in cotton or contribute to cotton yield. It protects the seeds only until boll opening, after which it withers. The fruit has no or little economic value (as compost). Cotton seeds with attached fiber represent the two major sinks of substantial economic value in the cotton crop. The cotton fiber is an elongated epidermal cell of the cotton seed coat; it is defined botanically as a trichome. Therefore, the two major sinks in seeds are: (1) the cotyledons of the seed embryo that store oil and protein; and (2) the secondary cell walls of the seed epidermal trichomes (cotton fibers) that store insoluble cellulose. Soluble sugars are not stored in any significant quantity in a mature cotton seed or fruit. Cotton seeds with their attached fiber represent all of the yield in the cotton crop. Therefore, cotton, as well as other fiber producing plants, differ significantly from tomato.
Increased total dry weight of vegetative parts of plants over-expressing SPS has
been shown in tomato leaves. In the same study, no change was observed in dry weight
of stems and root dry weight decreased (Galtier et al.,"Effects of Elevated Sucrose-
Phosphate Synthase Activity on Photosynthesis, Assimilate Partitioning, and Growth in
Tomato (Lycopersicon
Tomato leaves do not contain substantial fiber, being composed mainly of mesophyll
cells and conducting vascular tissue. The same plants were shown to sometimes have
Increased expression of SPS has been shown to exert other beneficial effects in
tomato and Arabidopsis. In both species, leaf starch storage is reduced in preference for
synthesis of sucrose. In both species, maximal rates of photosynthesis are enhanced, most
significantly in elevated C02 and saturating light (Galtier et al.,"Effects of Light and
Atmospheric Carbon Dioxide Enrichment on Photosynthesis and Carbon Partitioning in
the Leaves of Tomato (Lycopersicon esculentum L.) Plant Over-Expressing Sucrose
Phosphate Synthase,"J. Expt.
Thus, there exists a need for a method to control the level of synthesis of cellulose
in fiber producing plants, in particular cotton. There exists a need to be able to control
the yield and quality of fibers of commercial value, in particular cotton, under diverse
environmental conditions. A general need exists to be able to control the synthesis of
SUMMARY OF THE INVENTION The present invention generally relates to a method of controlling the cellulose synthesis in plants to optimize the level of production and quality of the products derived from the plants.
The invention includes the regulation in the cellulose content, thickness, or yield of any plant cell wall of agricultural or industrial use. Such cell walls include typical thin primary cell walls such as those that are digested in forage and those that exist in useful agricultural residues, for example beet root parenchyma cells remaining after sugar extraction that can be converted into thickening agents. Such cell walls include thick walls such as those of collenchyma and xylem parenchyma that can aid plant rigidity or contribute to yield and digestibility of forage or other agricultural products. Such cell walls also include secondary cell walls such as are commonly found in fiber.
In particular, the present invention provides a transgenic cotton plant that has an increased level of sucrose phosphate synthetase relative to a non-transgenic cotton plant.
The invention also provides a method of increasing the yield of a cotton plant by introducing into the cotton plant a chimeric DNA construct that alters the level of sucrose phosphate synthase activity in an amount sufficient to increase the seed and fiber yield of the cotton plant.
The present invention can also be used to increase the quality of cotton fiber produced from a cotton plant by introducing into a cotton plant a chimeric DNA construct that alters the level of sucrose phosphate synthase activity in an amount sufficient to increase the quality of the cotton fiber produced by the cotton plant.
The invention includes a method of increasing tolerance of photosynthetic
efficiency to cool night temperatures by introducing into a plant a chimeric DNA that
alters the sucrose phosphate synthase activity in an amount sufficient to increase tolerance
of photosynthetic efficiency to cool night temperatures.
In yet another embodiment, the invention provides a method of regulating the ratio of cellulose to other dry weight components of the plant by introducing into a plant a chimeric DNA construct capable of altering sucrose phosphate synthase activity in an amount sufficient to regulate the ratio of cellulose to other dry weight components of the plant.
The invention also provides a method of regulating the thickness of cell walls in a plant by introducing into a plant a chimeric DNA construct that alters sucrose phosphate synthase activity in an amount sufficient to regulate the thickness of cell walls.
In yet another embodiment, the invention provides a method of increasing the harvestable yield of fiber from a fiber containing plant by introducing into a plant a chimeric DNA construct that alters sucrose phosphate synthase activity in an amount sufficient to increase the harvestable yield of fiber from a fiber producing plant.
In yet another embodiment, the invention provides a method of increasing the harvestable yield of seed from a seed producing plant by introducing into a plant a chimeric DNA construct that alters sucrose phosphate synthase activity in an amount sufficient to increase the harvestable yield of seed from a seed producing plant.
In yet another embodiment, the invention provides a method of improving the quality of fiber from a fiber producing plant by introducing into a plant a chimeric DNA construct that alters sucrose phosphate synthase activity in an amount sufficient to regulate fiber quality. Such improvement may be exemplified by changes in length, strength, and weight per unit length.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the pathways of carbon assimilation, starch synthesis and catabolism, and sucrose synthesis. UDP-glucose pyrophosphorylase catalyzes the highly reversible reaction between glucose 1-phosphate (G-1-P) and UDP-glucose. Sucrose- phosphate synthase catalyzes the formation of sucrose-phosphate from UDP-glucose and fructose 6-phosphate.
Figure 2 shows the metabolic
Figure 3 is an amino acid alignment between SPS gene sequences from a number
of plant species.
Figure 4 is an amino acid alignment between the spinach leaf SPS gene sequence and a homologous sequence from Synechocystis.
Figure 5 is a histogram of fiber weight per seed, which shows elevation in all three transgenic lines. (Here and in all subsequent histograms, the error bars are standard deviations of the average. The average values are printed above each bar.) Figure 6 is a histogram of delinted seed weight per seed. It shows elevation in all three transgenic lines.
Figure 7 is a histogram of the ratio of fiber weight per seed and delinted seed weight per seed. It shows that these two yield parameters tend to increase in parallel, with a small preference for increased fiber weight in transgenic lines.
Figure 8 is a scatter plot of fiber weight per seed vs delinted seed weight per seed.
It shows that these two parameters are interdependent at the 50% level. (Here and with
all other scatter plots, R2 is the coefficient of determination calculated from the linear
regression line. Also, data points from parental C312 are labeled to their right, whereas
data point from the three transgenic lines are left unlabeled.) Note, however, that
Figure 9 is a histogram of fuzz fiber weight per seed. It shows elevation in two of three transgenic lines, and a decrease in one transgenic line.
Figure 10 is a histogram of micronaire, which shows elevation in all three transgenic lines.
Figure 11 is a scatter plot of micronaire vs fiber weight per seed showing that these two parameters are interdependent at the 60% level. This is sensible since fiber weight per seed depends on 3 factors: number of fibers, length of fibers, and fiber wall thickness. Of these 3 factors, micronaire would depend only on fiber wall thickness.
Note that this linear relationship also holds for C312, but the transgenics have higher values for fiber weight per seed and micronaire.
Figure 12 is a histogram of grams of force to break a single fiber (Tb; g). It shows
elevation in all transgenic lines.
Figure 13 is a histogram of elongation to break a single fiber (% of original fiber length). It shows elevation in all transgenic lines. However, note that Elongation is highest in transgenic line 13-3a, which, among the transgenics, had the lowest increase in grams to break. This suggests that these two factors are primarily determined by different fiber properties, as would be predicted in theory and is confirmed by the scatter plots below.
Figure 14 is a histogram of work to break a single fiber
Figure 15 is a scatter plot of grams of force to break a single fiber vs. micronaire.
The graph shows an interdependency for these parameters over all data points of
Both of these parameters would be expected to increase with a thicker fiber wall.
Figure 16 is a scatter plot of grams of force to break a single fiber vs. fiber weight
per seed. These parameters are interdependent at a level of
Figure 17 is a scatter plot of work to break a single fiber vs. micronaire. These parameters are interdependent at a level of 48%. The intermediary level of dependency compared to grams to break and elongation alone (See Figure 19) is reasonable for this composite factor.
Figure 18 is a scatter plot of work to break a single fiber vs. fiber weight per seed.
These parameters are interdependent at a level of 39%, which is similar to the dependence on micronaire (See Figure 17). As just described for Figure 16, this supports the hypothesis that increased fiber weight per seed is due in large part to increased fiber wall thickness.
Figure 19 is a scatter plot of elongation to break vs. micronaire. The graph shows that these parameters are not interdependent. Therefore, over-expression of SPS is predicted to enhance elongation by a mechanism independent of fiber wall thickness, which is consistent with theory.
Figure 20 is four overlayed scatter plots of photosynthetic rate vs. internal C02
concentration for parental C312 growing in the Phytotron. Empty symbols are for two
Figure 21 is four overlayed scatter plots of photosynthetic rate vs. internal C02
concentration for the transgenic line
Figure 22 is four overlayed scatter plots of photosynthetic rate vs. internal C02
concentration for the transgenic line 225-17a growing in the Phytotron. Empty symbols
are for two plants growing at
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of controlling the cellulose synthesis in plants to optimize the level of production and quality of the products, in particular fiber, derived from the plants.
The word"fiber"is often used to unify a diverse group of plant cell types that
share in common the features of having an elongated shape and abundant cellulose in
thick cell walls, usually, but not always, described as secondary walls. Such walls may or
may not be lignified, and the protoplast of such cells may or may not remain alive at
maturity. Such fibers have many industrial uses, for example in lumber and
manufactured wood products, paper, textiles, sacking and boxing material, cordage,
brushes and brooms, filling and stuffing, caulking, reinforcement of other materials, and
manufacture of cellulose derivatives. In some industries, the term"fiber"is usually
inclusive of thick-walled conducting cells such as vessels and tracheids and to fibrillar
aggregates of many individual fiber cells. Here the term"fiber"is used in its most
inclusive sense, for example including: (a) thick-walled conducting and non-conducting
cells of the xylem; (b) fibers of extraxylary origin, including those from phloem, bark,
ground tissue, and epidermis ; and (c) fibers from stems, leaves, roots, seeds, and flowers
or inflorescences (such as those
In a preferred embodiment, the invention provides a transgenic cotton plant wherein the transgenic cotton plant has an increased level of sucrose phosphate synthetase relative to a non-transgenic cotton plant. Table 1 shows the level of SPS activity from untransformed C312 plants and four transformed plant lines. All transformed plant lines show significant increases in SPS activity in both leaves and fiber.
Sucrose phosphate synthase plays a key role in the metabolic flux of carbon
within plant cells. Genes encoding sucrose phosphate synthase have been isolated and
sequenced from a number of plant species.
In addition to the known sequences of sucrose phosphate synthase, modifications
of the known sequences are also within the scope of the invention. Variations in the
The maximum activity of sucrose phosphate-synthase may be determined
colorimetrically according to the formation of sucrose-6-P (+ sucrose) from fructose-6-P
and UDP-glucose by the method as described in (Copeland,"Enzymes of Sucrose
Metabolism,"Methods in Plant
Unreacted hexoses or hexose phosphates were destroyed by immersion of tubes in a
boiling water bath for 10 min. After cooling to room temperature,
Alternatively, the activity of sucrose phosphate-synthase may be determined
spectrophotometrically according to liberation of uridine-5'-diphosphate detected by a
pyruvate-kinase coupling enzyme reaction as also described in (Copeland,"Enzymes of
In order to express the sucrose phosphate synthase in plants, transgenic plants carrying the gene encoding a sucrose phosphate synthase are produced by transforming a plant with a chimeric DNA construct that expresses sucrose phosphate synthase.
In order to express the sucrose phosphate synthase gene from the chimeric DNA, the construct should include a plant specific promoter. The promoter should ensure that the foreign gene is expressed in the plant. The promoter can be chosen so that the expression occurs only in specified tissues, at a determined time point in the plant's development or at a time point determined by outside influences. The promoter can be homologous or heterologous to the plant. Suitable promoters include e. g. the RUBISCO small subunit promoter, fiber-specific promoters, the promoter of the 35S RNA of the cauliflower mosaic virus described in U. S. Patent No. 5,034,322 (which is hereby incorporated by reference), the enhanced 35S promoter described in U. S. Patent No. 5,106,739 (which is hereby incorporated by reference), the dual S35 promoter, the FMV promoter from figwort mosaic virus that is described in U. S. Patent No. 5,378,619 (which is hereby incorporated by reference), the RI T-DNA promoter described in U. S.
Patent No. 5,466,792 (which is hereby incorporated by reference), the octopine T-DNA
promoter described in U. S. Patent No. 5,428,147 (which is hereby incorporated by
reference), the alcohol dehydrogenase 1 promoter (Callis et
Preferred promoters include the RUBISCO small subunit promoter, the 35S
promoters, fiber enhanced promoters, vascular cell enhanced promoters, stem cell
enhanced promoters, or seed enhanced promoters. Such promoters may ensure
Other promoters can be used that ensure expression only in specified organs, such as the leaf, root, tuber, seed, stem, flower or specified cell types such as parenchyma, epidermal, or vascular cells. One example of a tissue specific promoter is the RB7 promoter that is root specific (U. S. Patent No. 5,459,252, which is hereby incorporated by reference).
Such promoters may be used either alone or in combination to optimize over-expression in the most desirable set of tissues or organs.
Preferred cotton fiber-enhanced promoters include those of the cotton fiber-
expressed genes E6 (John et
Natl. Acad.
Preferred promoters enhancing expression in vascular tissue include the CAD 2
promoter (Samaj et al., Planta, 204: 437-443 (1998), which is hereby incorporated by
reference), the
Natl. Acad. Sci. USA, 95: 6619-6623 (1998), which is hereby incorporated by reference),
the PtX3H6 and PtX14A9 promoters (Loopstra et
Preferred promoters enhancing expression in stem tissue include pith promoters
(Datta, Theor. Appl. Genet., 97: 20-30 (1998) and Ohta et al., Mol. Gen. Genet., 225: 369-
378 (1991), which are hereby incorporated by reference), and the anionic peroxidase
promoter (Klotz et
Preferred promoters enhancing expression in seeds include the phas promoter
(Geest et
Truncated or synthetic promoters including specific nucleotide regions conferring
tissue-enhanced expression may also be used, as exemplified by identification of
regulatory elements within larger promoters conferring xylem-enhanced expression
(Seguin et
In one embodiment of the invention the chimeric DNA construct is stablely
integrated into the genome of the cotton plant. When a plant is transformed by
Numerous methods exist for transforming plant cells. The preferred methods include electroporation, Agrobacterium mediated transformation, biolistic gene transformation, chemically mediated transformation, or microinjection.
The vector described above can be
Genetics, 202 : 179-185 (1985), which is hereby incorporated by reference). The genetic
material may also be transferred into the plant cell using polyethylene glycol (Krens et al.,
Nature, 296: 72-74 (1982), which is hereby incorporated by reference).
Another approach to transforming plant cells with a gene that increases fiber and seed yield and fiber quality is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U. S. Patent Nos. 4,945,050,5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e. g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.
Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al., Proc. Natl. Acad. Sci. USA, 79: 1859-63 (1982), which is hereby incorporated by reference).
The DNA molecule may also be introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA, 82: 5824 (1985), which is hereby incorporated by reference). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids.
Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
Another method of introducing the DNA molecule into plant cells is to infect a
plant cell with
Heterologous genetic sequences can be introduced into appropriate plant cells, by
means of the Ti plasmid of A.
After transformation, whole transformed plants can be recovered. If transformed
seeds were produced directly, these can be selected by germination on selection medium
and grown into plants (Glough et al. The Plant Journal 16:
If protoplasts or explants were transformed, plants can be regenerated. Plant
regeneration from cultured protoplasts is described in Evans et
Callus tissue is formed and shoots may be induced from callus and subsequently rooted.
Alternatively, embryo formation can be induced in the callus tissue. These embryos
germinate as natural embryos to form plants. The culture media will generally contain
various amino acids and hormones, such as auxin and cytokinins. It is also advantageous
to add glutamic acid and proline to the medium, especially for such species as corn and
alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the
history of the culture. If these three variables are controlled, then regeneration is usually
reproducible and repeatable.
It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, species of sugarcane, sugar beets, cotton, forest trees, forage crops, and fiber producing plants. Regeneration is also possible in seed-producing plants including, but not limited to, maize, rice, wheat, soybean, rape, sunflower, and peanut.
After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the sucrose phosphate synthase resulting in enhanced seed yield and/or enhanced fiber yield and/or enhanced fiber quality. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
The present invention also provides seeds produced from the transgenic plant having increased synthesis of sucrose phosphate synthase.
In another embodiment, the invention provides a method of increasing the yield of
cotton plant by introducing into a cotton plant a chimeric DNA construct that alters
sucrose phosphate synthase activity in an amount sufficient to increase the yield of the
cotton plant. A chimeric gene may be introduced into plant cells or tissue. Transformed
cells are selected, usually by the use of a selectable marker. The transformed cells are
then used to generate a transformed plant (Fraley et al., Proc. Natl. Acad. Sci. USA,
79:
Preferred plants are cotton plants. The transformed plants may have an increase in the yield of cotton seeds or cotton fiber.
The present invention also provides a method of increasing the quality of cotton fiber produced from a cotton plant by introducing into a cotton plant a chimeric DNA construct that alters the sucrose phosphate synthase activity in an amount sufficient to increase the quality of the cotton fiber produced by the cotton plant.
The level of sucrose phosphate synthase may be increased by expressing factors
that increase the level of expression of the gene. Such factors may act on regulatory sites
controlling expression that are normally located near the sucrose phosphate synthase gene
or heterologous regulatory sites located near the gene in a chimeric construct.
Alternatively, the level of sucrose phosphate synthase may be increased by introducing a chimeric DNA construct that directly expresses a sucrose phosphate synthase.
Generally, the present invention can be used to change the ratio of cellulose to the dry weight of the whole plant or to the dry weight of plant components by introducing into a plant a chimeric DNA construct capable of altering sucrose phosphate synthase activity in an amount sufficient to change the ratio of cellulose to the dry weight of the whole plant or plant components. The change in cellulose can be observed in relation to total weight of the plant or fractionated parts of plants including, but not exclusively, starch, total cell walls, cell wall of fibers, particular organs such as stems, or cell wall components such as pectins, hemicelluloses, proteins, extractives, and lignin. The change in the ratio of cellulose to the fractionated parts of plants can be observed when the fractionated parts are considered alone or in any additive combination.
Changes in qualities as claimed in this invention refer to changes of at least 10% compared to a plant lacking the transgene. For example, the ratio of cellulose in cell walls may be changed from 20% to 18% or lower or 22% or higher. Such change compared to parental level could apply to all cell walls or any cell wall fraction of a plant.
In a preferred embodiment, the dry weight of cellulose may be increased so that its ratio to other dry weight components exceeds 40%. Such increase to exceed 40% could apply to wood, fibers, and other cellulose-rich cell walls such as collenchyma and thickened xylem parenchyma.
To accomplish certain changes, the level of sucrose phosphate synthase may be
decreased by expressing factors that decrease the level of expression of the gene. Such
factors may act on regulatory sites controlling expression that are normally located near
the sucrose phosphate synthase gene or heterologous regulatory sites located near the
gene in a chimeric construct. Alternatively, in anti-sense technology, the level of sucrose
phosphate synthase may be decreased by introducing a chimeric DNA construct that
contains the complementary
Sci. USA 96: 7398-7402 (1999), which is hereby incorporated by reference).
In yet another embodiment, the invention provides a method of increasing tolerance of photosynthetic efficiency to cool night temperatures by introducing into a plant a chimeric DNA construct capable of altering sucrose phosphate synthase activity in an amount sufficient to increase tolerance of photosynthetic efficiency to cool night temperatures.
The present invention can be used to regulate the thickness of cell walls in a plant by introducing into the plant a chimeric DNA construct that will change the sucrose phosphate synthase activity. In particular, the method can be used to increase the yield of harvestable fiber from any fiber producing plant.
In a preferred embodiment, the plant is a fiber producing plant. More preferred fiber producing plants are sugarcane, sugar beets, forest trees, forage crops, fiber producing plants, and seed producing plants.
In yet another embodiment, the present invention can be used to increase the harvestable yield of fiber from a plant. The invention may also be used to alter the quality of fiber isolated from the plant... Changes in sucrose phosphate synthase can change fiber strength, fiber length, or weight per unit length. Changes may either increase or decrease the strength, length or weight per unit length.
The present invention can be used to increase the yield of seed harvested from a seed producing plant by introducing into the plant a chimeric DNA construct that will increase the sucrose phosphate synthase activity.
The methods of the invention are broadly applicable and can be used in a wide variety of plants including cotton, forest trees, forage crops, beets, flax, hemp, jute, and other fiber-producing plants. They can also be used in seed producing plants including cotton, flax, wheat, rice, corn, soybean, Brassica sp. (e. g. rape), sunflower, safflower, peanut, palm, and other seed producing plants.
The methods of the invention are further described in the examples that follow.
EXAMPLES
Example 1-Materials and Methods
Most plants described were grown in one chamber at the Duke University
Phytotron: 360 ppm (normal) C02 ;
Other plants were grown in the Duke University Phytotron in 3 other chambers as
described except with the following changes: (a) 360 ppm
Other plants were grown in the Texas Tech University greenhouse: natural C02
and illumination; approximately
All open bolls were harvested from each plant from which seed and fiber parameters were evaluated. Lint fiber was removed from the seeds by hand-stripping.
Cotton seeds are covered with lint fiber (the long fiber used for textiles) and fuzz fiber
(short fibers used in various industrial applications). (Lint) fiber weight and fuzzy seed
weight from each plant was determined by weighing.
For plants for which stem weight was determined, any unopened bolls and leaves and petioles were removed. Above-ground stems were oven-dried and weighed.
The plant line used is a Coker 312 wild-type (untransformed parent) and four
transgenic lines. Transgenic plant lines, each known to represent separate transformation
events, are designated 13-3a, 225-17a, 40-4b, and 40-6a.
The number of individual plants grown in the Phytotron to yield average data for
each parameter (except for 40-6a-4) is indicated as Phytotron Plants (n) (Table 2). Line
40-6a-4, although it generally performed consistently with the other lines, was omitted
from fiber quality averages because it was represented by only one plant in the
Leaf and fiber RNA levels were determined by Northern analysis of the
The Boll # per Plant is the number of non-aborted bolls on each plant.
The Delinted Seed Weight per Seed (g) and (Lint) Fiber Weight per Seed (g)
(Table 2) are data derived from all open bolls of each plant at the time the experiment was
terminated. Under
Bulk (or bundle) fiber properties as determined by automated HVI and AFIS
testing are summarized in Tables 3 and 4. The fiber micronaire (by HVI) is a unitless
measurement that depends both on fiber maturity (or wall thickness determined by
secondary wall cellulose content) and fiber diameter.
Fiber bundle strength (by HVI) is expressed in units of
Fiber fineness (by AFIS) is expressed as (mTex). It represents the weight, in milligrams, of one kilometer of the fiber. One thousand meters of fibers with a mass of 1 milligram equals 1 millitex.
The fiber maturity ratio (by AFIS) is an expression of the degree of cell wall thickening (depending on secondary cell wall cellulose deposition). It is the ratio of fibers with a 0.5 (or more) circularity ratio divided by the amount of fibers with 0.25 (or less) circularity. (Fibers with thicker walls are less prone to collapse and remain more circular upon drying.) The higher the maturity ratio, the more mature the fibers are and the better the fibers are for dyeing.
The immature fiber content ("IFC%", by AFIS) is the percentage of fibers with
less than
Several different units are used as indicators of fiber length. Table 3 shows values for three of these as now described. Upper half mean ("UHM", by HVI) is the mean length of the longest one half of the fibers (weight biased). The fiber Uniformity Index ("UI", by HVI) expresses the ratio of the mean value (Mean Length) to the Upper Half Mean Length. It is a measure of the fiber length scatter within the population; if all fibers were the same length UI would equal 100%. Short Fiber Content ("SFC %", by HVI) is the percentage of fibers less than 1/2"long on a weight basis. HVI is thought to measure Short Fiber Content as determined by genetics only since the measurement does not impose additional potential fiber breaking stress.
Other fiber length indicators discussed in the text are as follows. The weight basis length ("L (w)" [in], by AFIS] is the average length of fibers calculated on a weight basis.
The number basis length ("L (n)"
Single fiber strength and elongation parameters derived from Mantis testing are
summarized in Table 5."Tb"
Detailed methods for particular experiments are included under the Examples.
Example 2-Summary of Results Demonstrating Increased Fiber and Seed Yield in
Transgenic Plants with Increased SPS Activity
Transgenic cotton plants with spinach SPS under the control of a constitutive
promoter showed foreign gene expression in the leaf and fiber as demonstrated by
Northern analysis. At the
Table 1
Characterization of Spinach SPS gene expression and
Total SPS Activity in Transgenic Plants
Over the first 9 weeks of growth in the
In the
Table 2
Yield Components of SPS Transgenic Plants Compared to
Parental C312 (at
Both cotton fiber and cotton seeds are valuable crops, the lint fibers for use in
textiles and other applications and the seeds as a source of oil and seed meal. In addition,
short fuzz fibers (also called linters) are harvested as a source of chemical cellulose,
among other uses. Increases were observed in number of bolls per plant, seed weight per
seed, fiber weight per seed, and fuzz fiber weight per seed. Boll number per plant
indicates overall capacity for production of seeds with attached fiber. Furthermore,
increased weight of seed and fiber per seed generates increased yield. Transgenic plants
over-expressing SPS achieve increased yield of two types of crops at the same time: seed
yield based primarily on storage of protein and oil and fiber yield based on storage of
cellulose. Therefore, plants that over-express SPS can be predicted to generate more
income per acre for the cotton producer based on crop yield alone. Coker 312 plants
over-expressing SPS can also be used for future transformations to help overcome any
potential yield drag from use of this old cultiver in genetic engineering. Seed and fiber
Increased Boll Number per Plant:
Three transgenic lines tested in the
Increased Fiber Weight per Seed:
Three transgenic lines tested in the
Increased Seed Weight per Seed:
Three transgenic lines tested in the
The ratio of Fiber Weight per Seed to Delinted Seed Weight per Seed in the
Increased Fuzz Fiber Weight per Seed:
Fuzz fiber weight per seed was obtained by subtracting the unit seed weight of
delinted seed from the unit seed weight of fuzzy seeds from the
Example 3-Summary of Results Demonstrating Increased Fiber Quality as Analyzed by Automated HVI and AFIS on Bulk Samples Many spinning properties of cotton depend on its properties as a bulk sample.
HVI and AFIS are automated systems that analyze these properties, yielding complementary information. These analyses show that the quality parameters of fiber produced by SPS transgenic plants are moving as a set into the premium quality range.
Fiber from SPS transgenic plants is longer, stronger, and more mature-all these features
are currently valued by the cotton processing and textile industries to make high quality
fabrics. Even under a stressful
Improvements Under 30/15°C, 360 ppm
Table 3
Fiber Quality Parameters of SPS Transgenic Plants Compared to Parental C312
(at
Table 4
Changes in Fiber Quality Parameters of SPS Transgenic Plants
(at
Micronaire. Three transgenic lines showed an average increase of 28% to attain an average micronaire of 4.72 (Fig. 10). Micronaire depends on secondary wall thickness and fiber diameter. It is desirable that increases in micronaire occur because of increased secondary wall thickness, not because of increased fiber diameter. The fiber diameter is estimated from the standardized relationship between Fiber Fineness and Fiber Maturity Ratio (Table 3) and found to be little-changed in transgenic lines. Both parental C312 and the transgenic lines had estimated fiber diameter between 16.5-17.0 µm.
Furthermore, a plot of Micronaire vs. Fiber Weight per Seed shows an interdependence at the 59% level (Fig. 11), supporting the existence of thicker walls in fibers of SPS transgenic plants. Other data on fiber strength, maturity ratio, and immature fiber content (see below) also support an increase in wall thickness of fiber from SPS transgenic plants.
Over 90% of the thickness of the cotton fiber wall is due to deposition of almost pure
Fiber Bundle Strength. Three transgenic lines showed an average increase of 12% to attain an average bundle strength of 30.3 cN/tex.
Fiber Fineness. Three transgenic lines showed an average increase of 8% to attain an average fineness of 180. Higher fiber fineness is traditionally undesirable because it is usually attributed to larger fiber diameter. However, since fiber of SPS transgenic plants has diameter approximately equal to parental C312 (see above), the increased fineness is likely attributable to increased fiber wall thickness yielding more weight per unit length.
Therefore, increased fineness of fiber from SPS transgenic plants is expected to be a neutral or positive fiber quality factor.
Fiber Maturity Ratio. Three transgenic lines showed an average increase of 7% to
attain an average maturity ratio of 0.95, which falls in the"above average"range (0.95-
1.00). This is superior to parental C312 with its average value of 0.89 in
Immature Fiber Content. Three transgenic lines showed an average decrease of
1.84% to attain an average of 5.61% immature fibers. Transgenic fibers are superior to
those of parental
Fiber length. Three transgenic lines showed an average increase in Upper Half
Mean length of 10% to attain average UHM of 1.14 inches. The three lines also have
more uniform fiber length, with average Uniformity Index increased 4.1% to attain
average UI of 87.2%. The three lines also have fewer short fibers, with average Short
Fiber Content by HVI decreasing 2.6% to attain average SFC% of 4.9 %. In addition to
data summarized in Tables 3 and 4, other AFIS parameters support increased fiber length
in fibers of SPS transgenic plants. For the average of three transgenic lines, L (w)
increases 7% to 1.06 inches, L (n) increases 9% to 0.96 inches, UQL (w) increases 6% to
1.19 inches,
Improvements Under Diverse Environmental Conditions:
Many fiber quality parameters were enhanced most for transgenic lines compared
to parental C312 in the
Micronaire. 4.65; 1.13x compared to the C312 average value.
Fiber Bundle Strength. 30 cN/tex ; 1.02x.
Fiber Maturity Ratio. 0.92,1.03x.
Immature Fiber Content. 6.69%; decreased 1.1%.
Length (n). 0.95 inches ; 1.08x.
Upper Quartile Length.
Fiber Uniformity Index. 87.7%; increased 1.3%.
Short Fiber Content (w) by HVI. 3. 77%; decreased
Short Fiber Content (w) by AFIS. 3.95%; decreased 1.75%.
Changes within each plant line are compared in average values for the quality
parameters of Micronaire, UHM,
Example 4-Summary of Results Demonstrating Increased Fiber Quality as Analyzed by Mantis Single Fiber Tests Cotton fibers with higher individual fiber strength are highly valued by the textile industry because they break less frequently during processing. Therefore, average fiber length can be maintained at a higher value throughout processing and higher quality fabrics can be manufactured with fewer defects. Increasing individual fiber strength is a major goal of the cotton industry.
Mantis tests to determine single fiber strength were run on 100 fibers (two independent groups of 50 fibers each) from at least 4 plants from each plant line.
Therefore, data in Table 5 are averages from at least 400 total fibers from each plant line.
Table 5
Single Fiber Strength of SPS Transgenic Plants Compared to Parental C312
(at
(HVI did not show any increase in Elongation % of transgenic lines compared to parental
The scatter plots in Figs. 15-19 show correlations between single fiber strength
parameters and Micronaire or Fiber Weight per Seed from the
Table 6
Coefficients of Determination (R2) from Linear Regression Plots
of Single Fiber Strength Parameters of Individual Plant Lines Plotted Against
Micronaire and Fiber Weight Per Seed
Also, the tendency for elevated Elongation in transgenic fibers is, as expected, independent of increased cellulose content of the fiber wall. (Elongation is highly dependent on the orientation of cellulose microfibrils within the fiber wall.) This point is emphasized by comparing line 13-3a with other transgenic lines.
Example 5-Photosynthetic Efficiency Under Cool Night Temperatures
Over-expression of SPS in the leaves increases tolerance to cool nights by
maintaining photosynthetic rates equal to warm-grown plants during the warm days
following a
Transgenic plants and parental C312 plants growing in the Phytotron were
assayed for photosynthetic efficiency between 7-14 weeks of age. The first fully
expanded leaf from the apex (judged by dark green color, shape, and size--the 3rd or 4th
leaf down) was clamped and assayed for photosynthetic efficiency using a ADC LCA-4
analyzer under variable internal C02 concentrations. Plants growing at 30/28°C were
assayed between 7-10 weeks of age and plants growing at
The graphs show photosynthetic rates over a range of internal C02 concentrations
for parental C312 (Fig. 21) and two transgenic lines,
The variability in plant age at the time of assay between
It is not yet known why plants over-expressing SPS fail to acclimate
photosynthesis in response to chilling as occurs in parental
Example 6-Shift of Metabolic Flux Toward Cellulose in Sink Cells
Tables 2 and 3 show that fiber properties depending on cellulose content,
including fiber weight/seed, micronaire, and fiber maturity ratio, increase in transgenic
plants when SPS activity is elevated both in the leaves and the fibers. Therefore, with
whole-plant analyses, one cannot judge whether these improvements are aided by
enhanced export of sucrose from the leaves to the fibers or enhanced synthesis of sucrose
in fiber (sink) cells, or both. Since cellulose synthesis has been proposed to use sucrose
as an obligatory substrate from which UDP-glucose is generated by the enzyme sucrose
synthase, SPS within sink cells can promote metabolic flux toward cellulose by one or
both of two mechanisms. SPS could resynthesize sucrose within sink cells because
Evidence that metabolic flux toward cellulose synthesis is enhanced in cellulose- storing sink cells (represented by cotton fibers) by over-expression of SPS was obtained from cotton ovules with attached developing fibers cultured in vitro. Cultured ovules/fibers are a non-photosynthetic system that uses external glucose in plant tissue culture medium as a carbon source to support metabolism required for seed and fiber maturation. Accepting that sucrose is an obligatory substrate for fiber cellulose synthesis, SPS synthesizes sucrose within tissue-cultured ovules/fibers supplied only with glucose.
SPS could also reuse the fructose released by the activity of sucrose synthase to
synthesize more sucrose. Positive effects of SPS over-expression observed in this system
are necessarily independent of photosynthesis. However, the substrate supply in this
tissue culture system is constant, implying that it is not possible to exclude enhanced
supply of sucrose due to enhanced SPS expression in leaves or decreased starch storage in
hypocotyls as also important in improvements observed in whole plants
Plants yielding the results in Table 7 were flowering in the greenhouse between
July and December. Ovules were dissected from flowers and cultured
The ovules of one flower were split between the
Comparison within one flower better controlled the variability that was observed in the
rates of cellulose synthesis on 21 DPA between cultures from different flowers of the
same plant line. Each test at each temperature included 12-18 ovules split between
three replicate dishes. Cultures were shifted from constant
Rates of respiration
From the
Results from parental C312 and 7 transgenic lines tested with good replication in parallel are shown in Table 7 with values considered higher than parental C312 shown in bold.
Table 7
Data Calculated From Rates of Cellulose Synthesis and Respiration
at
The data in Table 7 show that over-expression of SPS reduces
Correspondingly, the ratio of cellulose synthesis rate to respiration rate at
Example 7-Higher Rate of Weight Gain in Sink Cells (Cotton Fibers) During Primary and Secondary Wall Deposition The in vitro ovule/fiber culture system has provided direct evidence that over- expression of SPS in sink cells can lead to higher rates of fiber weight gain at both warm and cool temperatures by mechanisms independent of photosynthesis.
Ovules of transgenic and control C312 were cultured in vitro at constant
Table 8
Rates of Cellulose Deposition in Fibers Cultured
Line 40-6a and 40-17a are listed together and counted as one line because they
likely represent the same transformation event based on derivation from the same parent
callus and the same segregation ratio at T1.
Two of the transgenic lines
From replicated time-courses of fiber weight gain, absolute values of fiber dry weight were also compared at 15 DPA (end of primary wall deposition) and 30 DPA (after extensive secondary wall deposition) in the transgenic plant lines grown in the Phytotron, plus line 38-4a-1. Each data point is the average from three experiments, including fiber from a total of 24-30 ovules representing 15-24 flowers from 4-6 plants per line. The results are shown in Table 9.
Table 9
Weights of Fiber (mg/ovule) from in vitro Cultures
At 15 DPA, four transgenic lines show consistently greater weight gain than
parental C312 under
Example 8-Enhanced Stem Weight of Transgenic Cotton Plants The positive effects of SPS over-expression on cellulose synthesis in cotton fibers extends to other fibers. Fibers make up most of the weight of annual or perennial strong stems, such as are found in mature cotton plants. Therefore, the stem weight of cotton plants grown in the Phytotron and the Texas Tech greenhouse was determined (Table 10).
The conditions of the Texas Tech greenhouse were most similar to the Phytotron
Table 10
Normalized Values for Stem Weight, Diameter, and Height
(Average values for transgenic plants are normalized to the corresponding value for the
Coker 312 wild-type parent set to 1.00.)
In the Phytotron, stem weight increased by 10% or more in transgenic plants
compared to parental C312 in 11 of 15 cases (representing the matrix of plant lines x
chambers tested). The increases are particularly pronounced and consistent across three
chambers for line 40-6a-4, although there were few replicate plants in the Phytotron for
this line. Therefore, line 40-6a-4-3 was tested at the next generation (T3) in the Texas
Tech greenhouse with more replication in parallel with parental C312 and another
transgenic line,
Considering the main plant stem, excluding branches that were also weighed, as a right
cone with volume =
Example 9-Increased Stem Diameter in Multiple Lines of Transgenic Cotton
In addition to line 40-6a, some stems appeared bigger than others among
transgenic cotton plants growing in the greenhouse. However, these plants were of
different ages. To try to quantitate this observation, electronic calipers were used to
measure stem diameter approximately two inches above the soil line in all plants in the
greenhouse on 9/23/98 (which did not include all the plants of interest implicated by
previous studies). Date of planting was also recorded for each plant measured. By
analyzing values for the Coker
Plant Age Rate of Stem Diameter Increase
< 150 days 0.13 mm/day
160-200 days 0.10 mm/day
>210 days 0.06
Table 11
Transgenic Plant Lines with Enhanced Rates of
Stem Diameter Increase in the Greehouse
Example 10-Enhanced Conversion of Atmospheric C02 into Harvestable Crops,
Preferentially Cellulose-based Fiber
As shown in Table 12, comparison of data between the
Therefore, over-expression of SPS has a preferential effect on cotton fiber
production probably due to increasing sink demand of this cellulose-based sink. SPS
over-expression in fiber can, as previously demonstrated, preferentially increase
metabolic flux toward cellulose and fiber weight gain. Data supporting these conclusions
are shown in Table 12, which shows the percentage change in values of various
parameters when C02 was increased from 300 to 700 ppm under
Table 12
Percentage Change in Various Crop-Related Attributes
With Increase from 300 to 700 ppm C02 at
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.