Alteration of plant morphology by control of profilin...

Details Alteration of plant morphology by control of profilin... Alteration of plant morphology by control of profilin...

1998-04-17

2002-02-05

Benzion, Gary (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters plant part growth

C435S069100, C435S419000, C435S468000, C800S298000

Reexamination Certificate

active

06344601

ABSTRACT:

TECHNICAL FIELD
The present invention relates to the in vivo effects of profilin in plant cells. In particular, it has been found that manipulation of the level of expression of profilins in plants can lead to useful alterations in plant phenotype.
BACKGROUND OF THE INVENTION
Actin is the major cytoskeletal protein of most eukaryotic cells and actin filaments are often the primary determinants of cell shape and movement. The polymerization and depolymerization of actin filaments inside the non muscle cells are highly regulated, both spatially and temporally, to give the cell the ability to rearrange its cytoskeleton drastically within minutes in response to external stimuli or at a point of the cell cycle. In addition to the roles in cell shape and movements, the actin cytoskeleton in plants has been shown to play a role in cytoplasmic streaming, cytokinesis, cell expansion and plant development (Williamson, 1993; Meagher and Williamson, 1994). To achieve these dynamic rearrangements the cell relies on a variety of actin-binding proteins.
Profilin is an ubiquitous actin-monomer binding protein whose homologs are present in organisms ranging from fungi and amoebae through higher plants and mammals. They are low molecular mass (12-15 Kd), actin-binding proteins involved in the regulation of actin dynamics. Apart from actin-binding, profilins also bind to phosphatidylinositol 4,5-bisphosphate (PIP2)(Sohn et al., 1995), poly-L-proline (Bjorkegren et al., 1993) and a proline rich VASP (Reinhard et al., 1995; Haffner et al., 1995). Evidence suggests that profilin is a multi-functional protein (Haarer et al., 1990) which exerts both positive and negative effects on the actin polymerization (Theriot and Mitchison, 1993) and is involved in signal transduction pathways (Sohn and Goldschmidt-Clermont, 1994).
In vitro studies showed that profilin can lower the critical concentration for actin polymerization, stimulate polymerization and promote nucleotide exchange on actin monomers (for reviews see Theriot and Mitchison, 1993; Sohn and Goldschmidt-Clermont, 1994 and Sun et al., 1995). In vivo studies on profilin functions also yielded mixed results. Profilin mutations affected multiple actin-dependent processes in Drososphila (Verheyen and Cooley, 1994), blocked yeast cell budding (Haarer, 1990) and cytokinesis, as they cannot assemble the contractile rings, in
Schizosaccharomyces pombe
(Balasubramanian et al., 1994). Recent studies on
Dictyostelium discoideum
showed 60 to 70% increase in F-actin content at the rim below the plasma membrane when both profilins are deleted (Haugwitz et al., 1994). Microinjection of profilin into animal (Cao et al., 1992) or plant (Staiger et al., 1994) cells depolymerized actin. Profilin overexpression in stably transformed cells increased polymerization of actin at the cell periphery by prolonging cortical actin filament half-life (Finkel et al., 1994).
In addition to the above functions, profilin has been shown to be involved in at least two signal transduction pathways. They are the growth factor mediated receptor tyrosine kinase pathway and the Ras/G protein signaling pathway (for a review see Sohn and Goldschmidt-Clermont, 1994). Profilin has shown to bind PIP2 with high affinity and inhibits unphosphorylated phospholipase C (Goldschmidt-Clermont et al., 1990 and Drobak et al., 1994). This inhibition is overcome by growth factor mediated phosphorylation of phospholipase C (Goldschmidt-Clermont et al. 1991). The cyclase associated protein (CAP)/Srv2p is a component of the Ras/G protein signaling pathway and an yeast mutant of CAP was rescued by profilin, which suggested a possible role for profilin in this pathway (Vojtek et al., 1991). Profilin can bind to actin or PIP2 simultaneously with poly-L-proline proteins and proline rich sequences found in many proteins (Reinhard et al., 1995). This interaction with proteins that have proline rich sequences suggested a role for profilin in mediating membrane-cytoskeletal communications by physically attaching signaling proteins to plasma membrane and/or cytoskeleton.
In plants profilin has been identified and characterized in several species including maize pollen and tobacco (Staiger et al., 1993 and Mittermann et al., 1995), leaves and root nodules of
Phaseolus vulgaris
(Vidali et al., 1995) and
Arabidopsis thaliana
(Huang et al., 1996 and Christensen et al., 1996). Plant profilins have been shown to bind plant and animal actin In vitro (Valenta et al., 1993; Giehl et al., 1994 and Ruhlandt et al., 1995). Recently Perelroizen et al. (1996) reported that Arabidopsis profilin, like other profilins, can bind G-actin and promote assembly of actin filaments at the barbed end in vitro. In contrast to other profilins, Arabidopsis profilins do not accelerate the nucleotide exchange on actin. Despite these advances in plant profilin studies, there have been no reports so far on the in vivo functional analysis of profilins in plants.
SUMMARY OF THE INVENTION
We have found that over-production of profilin in plants or plant cells, preferably achieved by transformation of a plant or plant cell with a chimeric profilin gene that is highly expressed in plants, can cause increased cell elongation in growing plants. One of the results of this effect is a plant with a “tall” phenotype compared to the non-transgenic control plant. Another result is elongation of the root and root hair system of the plant.
We have also found that under-production of profilin, preferably achieved by reducing expression of native profilin in a plant (such as by the use of antisense RNA), can cause decreased cell elongation. One of the results of this is a “dwarf” phenotype.
It is also possible to direct over-expression or under-expression to specific tissues of the plant, such as root tissues. This results in a selective effect on the growth of that tissue. In one embodiment, a profilin coding sequence is linked to a root-specific promoter, resulting in elongation of roots, and particularly root-hairs. This increase in root surface area increases the plant's capacity to absorb water and nutrients, making it more drought-resistant, for instance, or less dependent on chemical fertilizers.
Control of the level of profilin production can also effect control of plant flowering time. It has been found that over-production of profilin by a plant leads to a delay in flowering time, while under-production of profilin promotes early flowering.
Such control of plant stature and flowering time has many applications in the creation of agricultural and horticultural varieties of plants.
DETAILED DESCRIPTION
The present invention takes advantage of the in vivo functions of plant profilins to effect changes in plant morphology, growth and flowering. It has been found that altering the level of expression of profilin in a plant cell leads to alterations in cell morphology and in cell elongation. At an organismic level, these changes can effect the growth habit of the plant, as well as effect the onset of flowering.
In an embodiment of the invention, a study was made of the in vivo functions of the
Arabidopsis thaliana
profilin-1 gene. Transgenic plants over- and under-expressing profilin-1 were generated by transforming wild type Arabidopsis plants with sense or antisense constructs under the control of a 35S promoter. Etiolated transgenic seedlings under-expressing profilin-1 exhibited overall stunted growth, short hypocotyl and at low temperature they were only 15 to 20% long when compared with the wild type plants. Light grown plants also showed the reduced growth and these plants flowered early. Light and electron microscopic observations of the hypocotyl surface and sections of the etiolated seedlings revealed that the surface of these seedlings were ruffled with some electron dense particle accumulation and enlarged epidermal, cortical and endodermal cells. The epidermal and cortical cells also showed defective vesicular transport with fibrous and electron dense particles and vesicles accumulating in the cytoplasm and close to the plasma m

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