Heat stable mutants of starch biosynthesis enzymes

Details Heat stable mutants of starch biosynthesis enzymes Heat stable mutants of starch biosynthesis enzymes

1999-05-14

2002-06-11

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers resistance to heat or cold

C536S023200, C536S023600, C800S284000, C800S298000, C800S320000, C800S320100, C800S320200, C800S320300, C800S276000, C435S419000, C435S468000, C435S412000, C435S194000

Reexamination Certificate

active

06403863

ABSTRACT:

BACKGROUND OF THE INVENTION
The sessile nature of plant life generates a constant exposure to environmental factors that exert positive and negative effects on its growth and development. One of the major impediments facing modem agriculture is adverse environmental conditions. One important factor which causes significant crop loss is heat stress. Temperature stress greatly reduces grain yield in many cereal crops such as maize, wheat, and barley. Yield decreases due to heat stress range from 7 to 35% in the cereals of world-wide importance.
A number of studies have identified likely physiological consequences of heat stress. Early work by Hunter et al. (Hunter, R. B., Tollenaar, M., and Breuer, C. M. [1977
] Can. J. Plant Sci.
57:1127-1133) using growth chamber conditions showed that temperature decreased the duration of grain filling in maize. Similar results in which the duration of grain filling was adversely altered by increased temperatures were identified by Tollenaar and Bruulsema (Tollenaar, M. and Bruulsema, T. W. [1988
] Can. J. Plant Sci.
68:935-940). Badu-Apraku et al. (Badu-Apraku, B., Hunter, R. B., and Tollenaar, M. [1983
] Can. J. Plant. Sci.
63:357-363) measured a marked reduction in the yield of maize plants grown under the day
ight temperature regime of 35/15° C. compared to growth in a 25/15° C. temperature regime. Reduced yields due to increased temperatures is also supported by historical as well as climatological studies (Thompson, L. M. [1986
] Agron. J.
78:649-653; Thompson, L. M. [1975
] Science
188:535-541; Chang, J. [1981
] Agricul. Metero.
24:253-262; and Conroy, J. P., Seneweera, S., Basra, A. S., Rogers, G., and Nissen-Wooller, B. [1994
] Aust. J. Plant Physiol.
21:741-758).
That the physiological processes of the developing seed are adversely affected by heat stress evident from studies using an in vitro kernel culture system (Jones, R. J., Gengenbach, B. G., and Cardwell, V. B. [1981
] Crop Science
21:761-766; Jones, R. J., Ouattar, S., and Crookston, R. K. [1984
] Crop Science
24:133-137; and Cheikh, N., and Jones, R. J. [1995
] Physiol. Plant.
95:59-66). Maize kernels cultured at the above-optimum temperature of 35° C. exhibited a dramatic reduction in weight.
Work with wheat identified the loss of soluble starch synthase (SSS) activity as a hallmark of the wheat endosperm's response to heat stress (Hawker, J. S. and Jenner, C. F. [1993
] Aust. J. Plant Physiol.
20:197-209; Denyer, K., Hylton, C. M., and Smith, A. M. [1994
] Aust. J. Plant Physiol.
21:783-789; Jenner, C. F. [1994
] Aust. J. Plant Physiol.
21:791-806). Additional studies with SSS of wheat endosperm show that it is heat labile (Rijven, A. H. G. C. [1986
] Plant Physiol.
81:448-453; Keeling, P. L., Bacon, P. J., Holt, D. C. [1993
] Planta.
191:342-348; Jenner, C. F., Denyer, K., and Guerin, J. [1995
] Aust. J. Plant Physiol.
22:703-709).
The roles of SSS and ADP glucose pyrophosphorylase (AGP) under heat stress conditions in maize is less clear. (AGP) catalyzes the conversion of ATP and &agr;-glucose-1-phosphate to ADP-glucose and pyrophosphate. ADP-glucose is used as a glycosyl donor in starch biosynthesis by plants and in glycogen biosynthesis by bacteria. The importance of ADP-glucose pyrophosphorylase as a key enzyme in the regulation of starch biosynthesis was noted in the study of starch deficient mutants of maize (
Zea mays
) endosperm (Tsai, C. Y., and Nelson, Jr., O. E. [1966
] Science
151:341-343; Dickinson, D. B., J. Preiss [1969
] Plant Physiol.
44:1058-1062).
Ou-Lee and Setter (Ou-Lee, T. and Setter, T. L. [1985
] Plant Physiol.
79:852-855) examined the effects of temperature on the apical or tip regions of maize ears. With elevated temperatures, AGP activity was lower in apical kernels when compared to basal kernels during the time of intense starch deposition. In contrast, in kernels developed at normal temperatures, AGP activity was similar in apical and basal kernels during this period. However, starch synthase activity during this period was not differentially affected in apical and basal kernels. Further, heat-treated apical kernels exhibited an increase in starch synthase activity over control. This was not observed with AGP activity. Singletary et al. (Singletary, G. W., Banisadr, R., and Keeling, P. L. [1993
] Plant Physiol.
102: 6 (suppl).; Singletary, G. W., Banisadra, R., Keeling, P. L. [1994
] Aust. J. Plant Physiol.
21:829-841) using an in vitro culture system quantified the effect of various temperatures during the grain fill period. Seed weight decreased steadily as temperature increased from 22-36° C. A role for AGP in yield loss is also supported by work from Duke and Doehlert (Duke, E. R. and Doehlert, D. C. [1996
] Environ. Exp. Botany.
36:199-208).
Work by Keeling et al. (1994, supra) quantified SSS activity in maize and wheat using Q
10
analysis, and showed that SSS is an important control point in the flux of carbon into starch.
In vitro biochemical studies with AGP and SSS clearly show that both enzymes are heat labile. Maize endosperm AGP loses 96% of its activity when heated at 57° C. for five minutes (Hannah, L. C., Tuschall, D. M., and Mans, R. J. [1980
] Genetics
95:961-970). This is in contrast to potato AGP which is fully stable at 70° C. (Sowokinos, J. R. and Preiss, J. [1982
] Plant Physiol.
69:1459-1466; Okita, T. W., Nakata, P. A., Anderson, J. M., Sowokinos, J., Morell, J., and Preiss, J. [1990
] Plant Physiol.
93:785-90). Heat inactivation studies with SSS showed that it is also labile at higher temperatures, and kinetic studies determined that the Km value for amylopectin rose exponentially when temperature increased from 25-45° C. (Jenner et al., 1995, supra).
Biochemical and genetic evidence has identified AGP as a key enzyme in starch biosynthesis in higher plants and glycogen biosynthesis in
E. coli
(Preiss, J. and Romeo, T. [1994
] Progress in Nuc. Acid Res. and Mol Biol.
47:299-329; Preiss, J. and Sivak, M. [1996] “Starch synthesis in sinks and sources,” In Photoassimilate distribution in plants and crops: source-sink relationships. Zamski, E., ed., Marcil Dekker Inc. pp. 139-168). AGP catalyzes what is viewed as the initial step in the starch biosynthetic pathway with the product of the reaction being the activated glucosyl donor, ADPglucose. This is utilized by starch synthase for extension of the polysaccharide polymer (reviewed in Hannah, L. Curtis [1996] “Starch synthesis in the maize endosperm,” In:
Advances in Cellular and Molecular Biology of Plants,
Vol. 4. B. A. Larkins and I. K. Vasil (eds.). Cellular and Molecular Biology of Plant Seed Development. Kluwer Academic Publishers, Dordrecht, The Netherlands).
Initial studies with potato AGP showed that expression in
E. coli
yielded an enzyme with allosteric and kinetic properties very similar to the native tuber enzyme (Iglesias, A., Barry, G. F., Meyer, C., Bloksberg, L., Nakata, P., Greene, T., Laughlin, M. J., Okita, T. W., Kishore, G. M., and Preiss, J. [1993
] J. Biol Chem.
268:1081-86; Ballicora, M. A., Laughlin, M. J., Fu, Y., Okita, T. W., Barry, G. F., and Preiss, J. [1995
] Plant Physiol.
109:245-251). Greene et al. (Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J., and Okita, T. W. [1996
] Proc. Natl. Acad. Sci.
93:1509-1513; Greene, T. W., Woodbury, R. L., and Okita, T. W. [1996
] Plant Physiol.
(112:1315-1320) showed the usefulness of the bacterial expression system in their structure-function studies with the potato AGP. Multiple mutations important in mapping allosteric and substrate binding sites were identified (Okita, T. W., Greene, T. W., Laughlin, M. J., Salamone, P., Woodbury, R., Choi, S., Ito, H., Kavakli, H., and Stephens, K. [1996] “Engineering Plant S

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