Transgenic plants with increased calcium stores

Details Transgenic plants with increased calcium stores Transgenic plants with increased calcium stores

2001-04-27

2004-06-22

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

C800S298000, C800S288000, C800S300000, C435S419000, C435S069800

Reexamination Certificate

active

06753462

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to transgenic plants, in particular, the present invention relates to transgenic plants expressing a calcium-binding protein that increases available calcium stores in the plant.
BACKGROUND OF THE INVENTION
Calcium plays an essential role in plant growth and development and is involved in multiple signal transduction pathways. Whereas cytoplasmic calcium concentrations are tightly regulated, higher levels of calcium are found in subcellular organelles (Gilroy et al., (1993)
J. Cell Science
106:453). Modulation of cytoplasmic calcium levels provides a rapid response to environmental stimuli and is achieved by a system of Ca
2+
-transport and storage pathways that include Ca
2+
buffering proteins in the lumen of intracellular compartments. The endoplasmic reticulum (ER), cell wall, and the vacuole contain high levels of calcium that could be released to the cytoplasm. Unlike animal cells, the majority of Ca
2+
in plant cells is found in the cell wall and vacuole, not in the ER (Bush, (1995)
Ann. Review Plant Physiol Plant Molec. Biol.
46:95). Except for the vacuole, which may not readily release calcium (Hirschi et al., (1999)
Plant Cell
11:2113), the availability of these stores for signaling has not been demonstrated. A voltage-gated, calcium-release channel has been identified in the endoplasmic reticulum (ER) of plants (Klusener et al., (1995)
EMBO J.
14:2708). This channel is responsive to mechanotransduction, suggesting that the ER calcium store may be an important component of signal transduction pathways in plants as well as animals (Klusener et al., (1995)
EMBO J.
14:2708). In plants, the major Ca
2+
storage protein in the ER is calreticulin (CRT) (Hassan et al., (1995)
Biochem. Biophys. Res. Commun.
211:54; Navazio et al., (1998)
Plant Physiol.
109:983).
Ca
2+
has long been recognized as an important second messenger responsible for mediating the activities of many environmental and endogenous signals. Cytosolic Ca
2+
concentrations often show significant changes in plant cells under the influence of various stress signals such as touch, cold or heat shock, wounding, anoxia, salinity, and hypoosmotic shock (Knight et al., (1991)
Nature
352:524; Knight et al., (1992)
Proc. Nat. Acad. Sci. USA
89:4967; Knight et al., (1996)
Plant Cell
8:489; Haley et al., (1995)
Proc. Nat Acad. Sci. USA
92:4124; Campbell et al., (1996)
Cell Calcium
19:211; Polisensky and Braam, (1996)
Plant Physiol.
11:1271; Subbaian et al., (1994)
Plant Physiol.
105:369; Lynch et al., (1989)
Plant Physiol.
90:1271; Bush, (1996)
Planta
199:89; Okazaki et al., (1996)
Plant, Cell and Environment
19:569; Takahashi et al., (1997)
Plant Physiol.
113:587). A stress-induced change in cytoplasmic calcium concentrations may be one of the primary transduction mechanisms whereby gene expression and biochemical events are altered to adapt plant cells to environmental stresses (Monroy et al., (1993)
Plant Physiol.
102:1227; Subbaiah et al., (1994)
Plant Physiol.
105: 369; Monroy and Dhindsa, (1995)
Plant Cell
7:321; Braam et al., (1996)
Physiol. Plant
98:909).
A variety of plant diseases and growth disorders causing substantial losses to horticultural crops have been attributed to calcium deficiency. Color breakdown in Anthurium spathes (Higaki et al., (1980)
J. Am. Soc. Hortic. Sci.
105;438; Higaki et al., (1980)
J. Am. Soc. Hortic. Sci.
105:441) can result in field losses of 50% and losses after shipments of up to 20%. Other conditions include but are not limited to tipburn in lettuce, cabbage and cauliflower (Goto & Takakura, (1992)
Trans. Am. Soc. Ag. Engineers
35;641; Barta & Tibbitts, (1986)
J. Am. Soc. Hortic. Sci.
111:413; Aloni, (1986)
J. Hortic. Sci.
61:509; Maynard et al., (1981)
Hotsci.
16:193), shoot-tip necrosis in potatoes, a physiological disorder found in normal microculture conditions, that makes the cultures useless for micropropagation or research (Sha et al., (1985)
J. Am. Soc. Hortic. Sci.
110:631); and blossom end rot in tomato (DeKock et al., (1980)
J. Sci. Food Agric.
33:509; Banuelos et al., (1985)
Am. Soc. Hortic. Sci.
20:894; Ho & Adams, (1994)
J. Hortic. Sci.
69:367). Addition of CaCO
3
does not remedy the problem in areas where additional factors such as soil salinity and pH are sub-optimal (Bower & Turk, (1946)
J. Am. Soc. Agron.
38:723; McLaughlin et al., (1993)
Can. J. For. Res. Rev. Can. Rech. For.
23:380; McCray et al., (1991)
Soil Use Manage
7:193; Francois et al., (1991)
Hort. Science
26:549) or where the condition results from localized deficiencies caused by uneven Ca
2+
distribution in tissues (Francois, et al, (1991)
Hort. Science
26:549; Ho & Adams, (1994)
J. Hortic. Sci.
69:367). Deficiencies may be exaggerated by high transpiration rates in a desert environment or a reduction in root pressure resulting from soil salinity (Francois, et al, (1991)
Hort. Science
26:549; Ho & Adams, (1994)
J. Hortic. Sci.
69:367). A temporary calcium deficiency of 8-10 days resulted in reduced stem growth and death of the apical meristem in tomato (Morand et al., (1996)
J. Plant Nutr.
19:115).
Calreticulin is a multifunctional calcium-binding protein that is highly conserved in eukaryotic cells (Michalak et al. (1998)
Biochem. Cell. Biol.
76:779; Michalak et al., (1999)
Biochem. J.
344 Pt. 2:281; Dresselhaus et al., (1996)
Plant Molec. Biol.
31:23; Krause & Michalak, (1997)
Cell
88:439). The conservation of CRT and the fact that CRT knockouts are lethal in mice (Mesaeli et al., (1999)
J. Cell. Biol.
144:857) suggest that CRT performs an essential function. In plants, CRT has been localized to the endoplasmic reticulum, Golgi, plasmodesmata, and plasma membrane (Borisjuk et al., (1998)
Planta
206:504; Hassan et al. (1995)
Biochem. Biophys. Res. Commun.
211:54; Baluska et al., (1999)
Plant J.
19:481). The protein includes a signal sequence and ER retention motif for ER localization, and also has a nuclear localization sequence. Although these localization sequences appear to be conserved across species, there is contradictory evidence for nuclear localization.
CRT has been shown to function as a chaperone in the ER (Peterson & Helenius, (1999)
J. Cell. Sci.
112:2775; Saito et al., (1999)
EMBO J.
18:6718; Denecke et al., (1995)
Plant Cell
7:391; Nauseef et al., (1995)
J. Biol. Chem.
270:4741; Qtteken & Moss, (1996)
J. Biol. Chem.
271:97; Crofts & Denecke, (1998)
Trends Plant Sci.
3:396). Other proposed roles include regulation of gene expression (Perrone et al. (1999)
J. Biol. Chem.
274:4640; signaling (Rauch et al., (2000)
Exp. Cell Res.
256:105), and serving as a calcium buffer (Mesaeli et al., (1999)
J. Cell. Biol.
144:857). In animal cells, calreticulin mRNA decreases during calcium depletion, along with resting and IP3-sensitive calcium pools (Mailhot et al., (2000)
Endocrinology
141:891). Persson et al. (in press) demonstrates that altered expression of calreticulin (CRT) altered Ca2+ uptake and release in ER-enriched membrane fractions. The data indicate that the pool of Ca2+ in the ER can be affected by altering expression of CRT.
CRT has three functional domains: a globular N-domain, a proline rich, high affinity, low capacity Ca
2+
-binding domain (the P-domain) and a highly acidic, low affinity, high capacity Ca
2+
-binding domain (the C-domain) (Michalak et al., (1992)
Biochem. J.
285:681). The P-domain shares considerable homology with the ER chaperone calnexin, which is also found in plants and functions as a chaperone. In addition, in Xenopus oocytes the P-domain has been implicated as the active region in Ca
2+
signal transduction (Camacho & Lechleiter, (1995)
Cell
82:765). The C-domain is a highly acidic region that has been shown to bind 20-50 moles of Ca
2+
/mole of protein and, thus, appears to be a major site of Ca
2+
storage within the ER (Michalak et al., (1992)
Biochem. J.
285:681). Calsequestrin, a calcium-binding protei

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