Patent Application: US-70590096-A

Abstract:
the present invention includes novel methods for increasing the seed productivity or accelerating the growth rate of a plant and plants produced by such methods . such plants have at least one cell transformed with an expression complex comprising a promoter operably linked to a gene encoding a pyruvate phosphate dikinase which is capable of converting pyruvate into phosphoenolpyruvate . the plants are made by transforming at least one plant cell with an appropriate expression construct , regenerating plants from one or more transformed plant cells and selecting at least one plant having the desired phenotype .

Description:
the cdna specific for the m . crystallinum ppdk encodes a precursor polypeptide of 103 kda processed to a 94 kda plastidic enzyme ( fiβlthaler 1993 ; fiβithaler et al . 1995 ). this cdna was introduced into a binary expression - vector ( bevan 1984 ; fig3 ). a control construct contains this cdna in reversed orientation ( δantisense ). this reversed cdna lacked 183 base pairs at its former 5 &# 39 ;- end . both ppdk cdnas were under control of the constitutive camv 35s - promoter . as a control the same vector containing β - glucuronidase ( vancanneyt et al . 1990 ) instead of ppdk was used . the constructs were transferred by agrobacterium tumefaciens to a random location of the plant genome . therefore , individual transformands are expected to vary in the degree of expression ( schell 1987 ). the control constructs ( δantisense - ppdk and β - glucoronidase ) were in all experiments very similar to the wildtype . further on , they will not always be mentioned . the heterologous ppdk and δppdk genes were transcribed in tobacco leaves ( fig4 ). sense - as well as δantisense mrna was detected . in comparison to the mrna level of ppdk in cam - performing leaves of m . crystallinum , the mrna level of the heterologous ppdk in the transformands was about 25 %. no ppdk mrna - signal could be obtained from the tobacco wildtype under the hybridization conditions used . ppdk protein could be detected in seeds as well as leaves of ppdk plants ( fig5 ). however , the protein amount of ppdk was much lower than expected on the basis of the northern analysis ( fig1 b ). in the wildtype or δantisense plants , no or only minor ppdk amounts could be detected . fig6 shows that the ppdk - overexpressing tobacco plants had higher ppdk activities as well ( ppdk : 125 - 166 % of the wildtype activity ). as expected , the degree of ppdk activity varied between individual transgenic plants . the β - glucuronidase control - transformand did not show increased ppdk activity . the ppdk activity of the wildtype was in the same range as reported by others ( edwards et al . 1980 ; kisaki et al . 1973 ). the evaluation of the productivity of the ppdk - overexpressing tobacco lines revealed that these plants grew faster ( fig7 ) and produced seeds about three weeks earlier than the wildtype . the data in fig8 demonstrate the effect of the additional ppdk on seed production . strikingly , all eleven lines analysed produced more seeds per seed capsule than the wildtype (= 100 %). line ppdk 6 reached the highest value with 174 %. this increase in productivity coincided with an increase in the ratio of the weights of seeds per seed capsule . while the wildtype seeds made up 50 % of the weight of the seed capsule , the seeds of the ppdk - overexpressing plants made up about 65 % of the weight of the seed capsule . in this respect , the most effective line was ppdk 2 with 77 %. in addition , the weight of the seed capsule itself was higher for most of the ppdk - overexpressing tobacco lines than for the wildtype . line ppdk 6 peaked here with 125 %. consistently , most of these lines produced more seeds altogether , too . ppdk 2 was the highest with 185 %. taken together , ppdk - overexpressing plants did not produce more seed capsules than the wildtype , but more seeds per seed capsule ( up to 74 % more ). the seeds produced by the ppdk - overexpressing tobacco were 90 % germinable , similar to the wildtype . tobacco seeds store mainly lipids ( about 48 % of the seed weight ), sugars and protein but no starch . no significant alterations were detected with regard to weight or composition of seeds of the ppdk - overexpressing plants ( fig9 ). tobacco seeds accumulate no starch but soluble sugars . the main storage compound were lipids ( 48 % of the seed weight ). free sugars and proteins made up only 8 % of the seed weight . the following conclusions can be drawn from the data : plants with additional plastidic ppdk grow faster . these plants produce more seeds than the wildtype . the increased seed productivity was due to heavier seed capsules and more seeds per seed capsule . the physiological basis of the increased growth rate and productivity of the ppdk - overexpressing tobacco is poorly understood at present , but is most likely due to refixation of carbon dioxide ( see fig1 ) produced by respiration . the additional plastidic ppdk produces the pepcase and refixation substrate phosphoenolpyruvate in increased amounts and so , remedies a energy waste and carbon loss bottle - neck . even the slightest measured increase in activity ( 125 %) initiated increased seed productivity and faster growth rates of the transformands ( fig6 and 8 ). the strong effects of ppdk on the growth rate and productivity of tobacco described here suggest that photosynthetic carbon assimilation is not limiting to growth and yield . it is surprising that a single enzymatic step can have such a dramatic effect on desirable traits in an agriculturally important plant . genetic engineering with a plastidic ppdk to produce additional pep should also improve other plants in the parameters described above . aoyagi k . and bassham j . a . 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