Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-11-24
2004-01-13
Bui, Phuong T (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S419000
Reexamination Certificate
active
06677506
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates in general to DNA molecules encoding a new polypeptide of the 11-12 transmembrane domain transporter family having a Mg
2+
- or Zn
2+
-proton exchange activity, expression vectors comprising them, plant cells transformed thereby and transgenic plants expressing same.
In all living organisms, cellular functions require a fine homeostasis of various ions and nutrients, including Mg
2+
and Zn
2
+. Mg
2+
is required for the function of manv enzymes (e.g., phosphatases, ATPases. RNA polymerases). Zn
2+
plays both a functional (catalytic) and structural role in several enzyme reactions, and is involved in the regulation of gene expression by zinc-finger proteins. Both Mg
2+
and Zn
2+
are essential for the structural integrity of ribosomes. In plants, Mg
2+
is also an essential component of chlorophyll, and regulates the activity of key chloroplastic enzymes.
Multicellular organisms have to balance not only their Mg
2+
and Zn
2+
intake and intracellular compartmentalization. but also the distribution of these ions to various organs. The movement of ions through membrane barriers is mediated by specialized proteins—channels, transporters or ATPases. Thus far, genes encoding Mg
2+
transporters have been cloned only from bacteria and yeast. The bacterial MgtA and MgtB Mg
2+
transport proteins are P-type ATPases (Hmiel et al., 1989). Mg
2+
is also transported by the bacterial CorA and mgtE proteins (Smith et al., 1993; Smith et al., 1995), but the molecular mechanism of Mg
2+
mobilization by these proteins is not known. Among the Zn
2+
transport proteins whose genes have been cloned, the bacterial ZntA (Rensing et al., 1997) is a P-type ATPase. Zn
2+
is also transported by the yeast ZRT 1,2 (Zhao and Eide, 1996a; Zhao and Eide, 1996b), transporters, but the molecular mechanism of Zn
2+
transport by these proteins is also unknown. A mammalian protein designated DCT1 (Gunshin el al., 1997), which belongs to the Nramp family of macrophage proteins, was suggested to be a symporter of protons with various divalent metal cations, including Fe
2+
and Zn
2+
, but it was not able to symport Mg
2+
ions.
Little is known about transport proteins that control. Mg
2+
and Zn
2+
homeostasis in plants. Ions absorbed into the cytosol of root cells diffuse towards the vascular cylinder through plasmodesmata and reach the xylem parenchyma cell layer, which border the xylem vessels. The xylem parenchyrna cells were suggested to play a key role in ion secretion into the xylem (xylem loading), and in the release of ions from the xylem (unloading). These processes require transport through the plasma membrane of the xylem parenchyma cells, but the proteins mediating xylem loading and unloading of Mg
2+
and Zn
2+
are not known. Unloaded Mg
2+
and Zn
2+
subsequently enter the surrounding cells through unknown transport proteins. The molecular mechanisms of phloem loading and unloading with Mg
2+
and Zn
2+
have also not been elucidated. Intracellularly, the vacuole is considered the main organelle mediating Mg
2+
homeostasis in the cytosol and the chloroplast. Vacuolar Mg
2+
is also important for the cation-anion balance and turgor regulation of cells. The activity of a Mg
2+
/H
+
antiporter was identified in lutoid (vacuolar) vesicles of
Hevea brasiliensis
(Amalou et al., 1992; Amalou et al., 1994) and in vacuolar membranes from roots of
Zea mays L
. (Pfeiffer and Hager, 1993), but cloning of the corresponding genes has not been reported. The
Hevea brasiliensis
transporter was indicated to be electroneutral, and to be capable of transporting also Zn
2+
cations. In Zn
2+
tolerant species, tolerance is achieved mainly through sequestering Zn
2+
in the vacuoles, but the transport mechanism is not known.
The progressive salinization of irrigated land threatens the future of agriculture in the most productive areas of our planet. Increasingly, intensive irrigation practices are resulting in secondary salinization of agricultural soils. Even water of good quality may contain 100-1000 g salt/m
3
. With an annual application of 10,000 m
3
/ha, between 1 and 10 t of salt are added to the soil. As a result of transpiration and evaporation of water, soluble salts further accumulate in the soil. Since crop productivity of irrigated land in many areas is much higher than of non-irrigated land, the coincidence of irrigation and salinization threatens current agricultural productivity. It has been estimated that 10×10
6
ha per annum of irrigated land are abandoned due to salinization and alkalization. For example, large areas of the Indian subcontinent have been rendered unproductive by salt accumulation and poor water management; in Pakistan, about 10 million of 15 million hectares of canal-irrigated land are becoming saline. Worldwide, about 33% of the irrigated land is affected by salinity, and presumably more land is going out of irrigation due to salinity than there is new land coming into irrigation.
Salinity problems occur also in non-irrigated croplands and rangelands either as a result of evaporation and transpiration of saline underground water or due to salt input from rainfall. The saline areas of the world consist of salt marshes of the temperate zones, mangrove swamps of the subtropics, and their interior salt marshes adjacent to salt lakes. Saline soils are abundant in semiarid and arid regions, where the amount of rainfall is insufficient for substantial leaching.
Soluble salts accumulating in the soil must be removed periodically by leaching and drainage. But even when proper technology is applied to the soils, they contain salt concentrations which often impair the growth of crop plants of low salt tolerance. Most crop species and cultured woody species either have a relatively low salt tolerance, or their growth is severely inhibited even at low substrate salinity. Salinity is the major nutritional constraint on the growth of wetland rice.
In saline soils, NaCl is usually the dominant salt. There are three major constraints for plant growth on saline substrate (Marschner, 1995, p. 662): (1) water deficit (‘drought stress’) arising from the low (more negative) water potential of the rooting medium; (2) ion toxicity associated with the excessive uptake of mainly Cl
−
and Na
+
; (3) nutrient imbalance, caused by depression in uptake and/or shoot transport and impaired internal distribution of mineral nutrients, and calcium in particular.
In many fruit trees and herbaceous crop species, ion toxicity is characterized by growth inhibition and injury of foliage (marginal chlorosis and necrosis on mature leaves). These phenomena occur even at low levels of NaCl salination, under which water deficit is not a constraint. Many plant species such as citrus and leguminous suffer from Cl
−
toxicity. The species that suffer most from Na
+
toxicity are graminaceous such as wheat, sorghum, and rice. Many crop species with relatively low salt tolerance are typical Na
+
excluders, and are capable at low and moderate salinity levels of restricting the transport of Na
+
into the leaves where it is highly toxic in salt sensitive species. The causes of salt toxicity in cells are inhibition of enzyme reactions and inadequate compartmentalization between cytoplasm and vacuole. There is also increasing support for the hypothesis of Oertli (1968) of salt accumulation in the leaf apoplasm as an important component of salt toxicity, leading to dehydration and turgor loss and death of leaf cells and tissues.
The mechanism of adaptation of plants to saline substrates is based on the principle that salt tolerance can be achieved by salt exclusion or salt inclusion. Differences in the capacity for Na
+
and Cl
−
exclusion exist between cultivars of different species. For example, the higher salt tolerance o
Galili Gad
Hilgemann Donald W.
Inze Dirk
Shaul Orit
Van Montagu Marc
Bui Phuong T
Collins Cynthia
G. E. Ehrlich Ltd.
Yeda Research and Development Co. Ltd.
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