Plastid-targeting nucleic acid sequence, a novel...

Details Plastid-targeting nucleic acid sequence, a novel... Plastid-targeting nucleic acid sequence, a novel...

1999-08-16

2002-12-03

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters carbohydrate production in the plant

C800S287000, C800S298000, C800S317200, C800S320300, C800S320100, C800S320000, C800S317400, C800S320200, C800S317300, C800S312000, C435S070100, C435S419000, C435S430000, C435S320100, C435S252300, C435S201000, C536S023600

Reexamination Certificate

active

06489540

ABSTRACT:

The precise mechanisms by which starch is synthesised and degraded in plants are unknown, despite the isolation and characterisation of a number of enzymes that are presumed to be involved in the process.
Starch is accumulated in the chloroplasts of leaves during the day and is used to supply the needs of the plant for energy and biosynthesis during the night. The mode by which this so-called transient starch is mobilised is not fully understood, but must involve the co-ordinated regulation of synthetic and degradative enzyme activities. In leaf tissues the main degradation pathway is thought to involve phosphorolytic and hydrolytic activities, especially &agr;-glucosidase (E.C. 3.2.1.3) (Nielson and Stitt, 1997).
Starch is also accumulated in the amyloplasts in storage organs such as seeds, fruit and tubers. In this case starch is stored over longer periods of time and mobilisation of the starch is accompanied by degeneration of the storage organ tissues and increases in amylolytic and phosphorolytic activities. However, there is evidence to suggest that turnover of starch is also occurring in the amyloplasts of the storage organ (Sweetlove et al, 1996). This again requires the co-ordinated regulation of the synthetic and degradative enzyme activities.
Chloroplasts and amyloplasts are both derived from proplastids and therefore have many characteristics in common besides being the site of starch synthesis in leaves and storage organs respectively; chloroplasts can be converted to amyloplasts and other types of plastid (Thomson and Whatley, 1980).
Starch is a mixture of two polysaccharides: amylose which is a linear chain of glucosyl units linked by &agr;-1,4-glycosidic bonds; and amylopectin which is made up of many linear chains of &agr;-1,4-polyglucans which are joined together by &agr;-1,6 glycosidic bonds.
Enzymes involved in the synthesis of starch are ADPG pyrophosphorylase (E.C. 2.7.7.21), starch synthase (E.C. 2.4.1.21) and branching enzyme (E.C. 2.4.1.18). ADPG pyrophosphorylase is responsible for supplying the substrate ADPG, this molecule serving as the donor of glucose monomers which are linked together by the concerted action of starch synthases (&agr;-1,4 bonds) and branching enzymes (&agr;-1,6 bonds).
It is thought that the insoluble, crystalline structure of starch grains is formed by the close packing of the extended helical, branched amylopectin molecules, with the linear amylose molecules filling any spaces.
A range of starch-degrading enzyme activities has been reported including &agr;-amylase (E.C. 3.2.1.1), isoamylase (E.C. 3.2.1.68), &bgr;-amylase (E.C. 3.2.1.2), &agr;-glucosidase (E.C. 3.2.1.3), starch phosphorylase (E.C. 2.4.1.1) and disproportionating enzyme (E.C. 2.4.1.25). Many of these enzyme activities exist in multiple forms in plants and some are thought to be involved in the synthesis of starch. All probably take part, to some extent, in the starch mobilisation process, however their exact roles and possible interactions are yet to be determined. The difficulties in attributing roles for the different enzymes is best exemplified by reference to two of the enzyme activities which are thought to. be the major contributors to starch breakdown in plants: starch phosphorylase and amylase.
Starch phosphorylase catalyses the reversible release of glucose-i-phosphate from &agr;-1,4-glucans. Two forms of starch phosphorylase are found in plant tissues: Pho1, or the L-type, is located inside plastids and has a high affinity towards maltodextrins; Pho2, or the H-type, is cytosolic and has high affinity to large, highly branched polyglucans such as glycogen. Although the plastidic Phol enzyme would be a likely candidate to be involved in the mobilisation of starch, antisense inhibition of the leaf enzyme activity had no effect on the starch accumulation in leaves of transgenic potato plants (Sonnewald et al., 1995). In another study, antisense inhibition of the cytoplasmic Pho2 had an influence on the sprouting behaviour of transgenic potato tubers, but had no effect on the starch accumulation and degradation (Duwenig et al., 1997).
There are two major groups of amylase both of which hydrolyse &agr;-1,4-glucosidic linkages in amylose and amylopectin: &agr;-amylase acts randomly on non-terminal linkages, whereas &bgr;-amylase acts to release maltose units starting from the non-reducing end of the polyglucan chain. The subcellular location of &agr;-amylase in the apoplastic space of plant cells is thought to reflect the fact that the enzyme is normally secreted. However, in a number of plants such as rice (Chen, et al., 1994) and sugar beet (Li, et al., 1992) the enzyme is also located inside chloroplasts and amyloplasts, despite the finding that the signal sequences at the amino-terminus of a number of &agr;-amylase proteins are characteristic for translocation of protein across the ER membrane rather than the plastid membrane (Chen et al, 1994). In a study where the promoter and signal sequence of a rice &agr;-amylase gene was fused to the bacterial GUS gene and introduced into rice, tobacco and potato using Agrobacterium-mediated transformation (Chan et al., 1994), it was demonstrated that the expressed GUS fusion protein was first transported to the endoplasmic reticulum and then exported into the culture medium of suspension cultures made from transgenic cells. It has been shown in a number of studies that &agr;-amylase will degrade native starch molecules.
In contrast, in vitro studies have shown that &bgr;-amylase will not degrade native starch granules without prior digestion of the granule with other enzymes. Mutants of rye (Daussant et al., 1981) and soybean (Hildebrand and Hymowitz, 1981) that lack active &bgr;-amylase or contain only traces of activity, respectively, apparently show normal growth and development. In addition, transgenic Arabidopsis plants in which the levels Of &bgr;-amylase have been greatly reduced, do not show severe growth defects (Mita et al., 1997). Attempts to define the precise physiological role of &bgr;-amylases in plants have been hampered by inconclusive data concerning subcellular location. Although one study (Kakefuda et al., 1986) reported the presence of two, &bgr;-amylases in pea chloroplasts, most studies involving species such as
Vicia faba,
barley, wheat, soybean, sweet potato and pea have concluded that most, if not all, &bgr;-amylase activity is extrachloroplastic (Nakamura et al., 1991). This view is supported by the fact that all &bgr;-amylase genes cloned to date encode proteins that lack amino-terminal chloroplast transit peptide sequences.
In cereals, three types of &bgr;-amylase have been described: an endosperm-specific form that accumulates during caryopsis maturation; a form that is synthesised de novo in aleurone cells of rice and maize during germination (Wang et al., 1996; 1997); and a &bgr;-amylase which is ubiquitous in vegetative organs. In Arabidopsis, the ubiquitous form accounts for approximately 80% of the total starch-degrading activity of rosette leaves. In common with all other &bgr;-amylase genes cloned to date, the gene for the ubiquitous Arabidopsis &bgr;-amylase does not encode a protein with a subcellular targeting signal, thus the enzyme is likely to be located in the cytosol.
The findings from a number of studies that the degradative activities can be removed without an adverse effect on the viability of the plant, plus the subcellular location of starch degrading enzymes outside the plastid, is surprising. The apparent absence of a plastid-localised &bgr;-amylase activity is especially surprising in light of the fact that the expected major end-product of &bgr;-amylase activity, namely maltose, has been identified as a product of starch degradation in isolated chloroplasts (Peavey et al., 1977). More recently, it has been shown that both glucose and maltose are exported from isolated cauliflower bud amyloplasts during the process of starch mobilisation (Neuhaus et al., 1995).
The ability to manipulate the amount of starch in the plastids of leaves or storage organs would be

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