WO2013005211A2

WO2013005211A2 - Boron complexing plant materials and uses thereof cross-reference to related applications

Info

Publication number: WO2013005211A2
Application number: PCT/IL2012/050233
Authority: WO
Inventors: Menachem Moshelion; Nava MORAN; Netta Li LAMDAN; Ziv ATTIA
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2011-07-05
Filing date: 2012-07-04
Publication date: 2013-01-10
Also published as: WO2013005211A3

Abstract

This invention relates to methods for reducing the boron concentration of a water source, solid supports for effecting the same, methods for increasing boron tolerance in a plant and seeds and transgenic plants produced in accordance with the same. The methods of this invention include providing conditions whereby the plant exhibits increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof, or providing conditions whereby a shoot in said plant contains intracellular complexes of malic acid, fructose, glucose, citric acid or a combination thereof with boron, in turn reducing toxicity of boron to the plant, thereby being a method of increasing boron tolerance in the plant. Methods of reducing soil boron content are also described.

Description

BORON COMPLEXING PLANT MATERIALS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of United States Provisional Application Serial Number 61/504,432 filed on July 5^th, 2011, hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[002] This invention relates to the field of botany and more specifically to plant materials, the reduction of the concentration of boron in water via the use of same, in particular in desalinated water and other uses thereof.

BACKGROUND OF THE INVENTION

[003] Boron (B) is a small, water soluble molecule, found in sea water at concentrations of 3-6 mg/L (3-6 ppm, or, roughly, 0.3-0.6 mM). Elsewhere, in the ground water and in the soil, boron concentrations vary widely and depend on the surrounding geology and wastewater discharges.

[004] Boron (B) is thought to be an essential microelement for plant growth. The involvement of boron in normal vegetative and especially in reproductive growth stages has been shown, although its biochemical role in these stages in not fully understood. Under normal growth conditions in land plants 90% of the boron is found in the cell wall, cross-linking pectin molecules in Rhamnogalacturonan (RG-II) complexes. Boron enters the root as boric acid (B(OH)₃), moves up with transpiration stream and is deposited in stems and leaves of vascular plants proportionally to the transpiration rate, though modifiable by environmental factors. In the apoplast (at a pH about 5.5), more than 99.95% of boron is in the form of boric acid (pKa 9.24) and <0.05% is in the form of borate (tetrahydroxyborate: B(OH)₄ ^") ions. In the cytoplasm (at pH about 7.5) more than 98% of boron is in the form of boric acid and less than 2% in the form of borate ion.

[005] In certain plant species, boron complexation with two molecules of alcoholic sugars (e.g. fructose, sorbitol, mannitol, dulcitol) via a pair of cis-hydroxyls has been suggested to impart mobility to boron in the phloem, allowing this generally stationary element to move to sinks. Boron complexation with cis-hydroxyls has been also suggested as a mechanism of averting boron toxicity (i.e., as a mechanism of cellular boron tolerance) in some Brassicaceae.

[006] While long-term studies in mice and rats indicate that boric acid and borax are not tumorigenic or genotoxic, short- and long-term oral exposures to boric acid or borax in laboratory animals have demonstrated that the male reproductive tract is a consistent target of boron toxicity. Testicular lesions have been observed in rats, mice and dogs given boric acid or borax in food or drinking-water. Developmental toxicity has been demonstrated experimentally in rats, mice and rabbits. The world health organization therefore set a guideline value for boron level in products of desalination (drinking water) to be 2.4 mg/L (i.e., 0.24 mM). As of 2004, the European Union issued a drinking water Directive, setting the limit within drinking water to have at most a boron concentration of 1 mg/L, i.e., 0.1 mM.

[007] In most plants the concentration range between boron deficiency (0.03-0.1 mmol B kg^"1 FW, i.e., roughly 0.3 to 1 mg B kg^"1 FW) and toxicity (> 0.1 mmol B kg^"1 FW in sensitive plants), is very narrow. Boron toxicity can be found in soils of the Middle East, South Australia, on the west coast of Malaysia, the southern beaches in Peru, northern Chile and India, where the highest concentrations of boron are found in evaporated marine soils. In addition to these naturally spreading boron-laden land areas, agricultural soils, particularly in semi-arid areas, are increasingly contaminated by boron through irrigation by treated wastewater or desalinated sea or saline water. These water sources abound in boron, and its removal is extremely costly, and therefore still largely impractical. With time, boron toxicity renders these agricultural areas limited to boron-resistant crops, and eventually they become non-arable.

[008] For example, around 10% of the investigated ground water resources in the Mediterranean basin have boron levels exceeding the established European Union Drinking Water Directive, many of them, like in southern coastal aquifer that is shared between Israel and the Gaza Strip, in Greece, in Cyprus and Italy, mostly from natural (geological) sources. In these areas, decreasing boron usage and its flow to wastes did not solve the problem.

[009] While 0.1 milimolar (1 mg/L) concentrations are considered safe for humans and various animals, boron is already toxic to arthropods and to some plants and diminishes agricultural crops. Among the most boron-sensitive plants (0.5-1 mg boron/ L, i.e., 0.05-0.1 mM) are blackberry, citrus, peach, cherry, plum, grape, cowpea, onion, garlic, sweet, potato, wheat, barley, sunflower, sesame, strawberry.

[0010] A visible symptom of boron toxicity in a variety of plant species is necrosis along leaf margins. In many plants in which boron is mobile in the phloem toxicity symptoms may rather occur in the fruit (such as gummy nuts and internal necrosis) and in other sink tissues (such as necroses in buds and young stems). Besides injury to plants, boron toxicity is evident as decreased fruit yield of crops (for example, tomato fruit yield decreased by 3.7% with each additional increase of 0.1 mM B in the soil solution above a threshold of 0.53 mM B).

[0011] Physiological effects of boron toxicity include reduction in cell division in the root, reduction in elongation rates of the root and the shoot, decreased chlorophyll level in the leaf, reduction in photosynthesis and stomatal conductance, excess production of lignin and suberin, decreasing of external root zone acidification, increase of membrane leakiness and increased fatty acids oxidation. These findings indicate the involvement of multiple processes which contribute to the overall boron toxicity. The formation of complexes with polyols, mentioned above as possibly contributing to increased boron mobility within the phloem, may constitute also the chemical basis of boron toxicity (in particular the complexation with ribose, which limits the availability of ribose - a moiety within many important molecules).

[0012] Some plants, such as various crop cultivars or halophytes, are significantly more resistant to boron than other members of their family but the mechanism whereby such plants are resistant to boron is unknown.

SUMMARY OF THE INVENTION

[0013] In one embodiment, this invention further provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water.

[0014] In some embodiments, this invention further provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water

[0015] In some embodiments, according to this aspect, reference to "increased concentration of malic acid, fructose, citric acid or a combination thereof" refers to a concentration that is higher than that present in a plant member of a related plant family.

[0016] In some embodiments, such methods referring to an "increased concentration of malic acid, fructose, citric acid or a combination thereof" refer to a concentration of malic acid, fructose and glucose ranging from at least 2 mmol kg^"1 FW to 20 mmol kg ln some embodiments, according to this aspect, the plant material is fresh or dried. In some embodiments, according to this aspect, the plant material is derived from a Thellungiella salsuginea plant, which is also known in the art as being a Thellungiella halophila plant. In some embodiments, according to this aspect, the water is contacted with a solid support comprising said plant material and in some embodiments, the solid support includes a column or other filtering device. [0017] In some embodiments, according to this aspect, a plant exhibiting increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof is grown by hydroponics within the water, as part of such method.

[0018] In some embodiments, this invention provides a solid support comprising a plant material exhibiting an increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family member, for a period of time sufficient to promote boron accumulation within said plant material. In some embodiments, according to this aspect, the plant material is derived from a Thellungiella salsuginea plant. In some embodiments, according to this aspect, the solid support is a column or filtering device.

[0019] In some embodiments, this invention provides for a method for reducing the boron concentration in soil, said method comprising contacting soil containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family member, for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said soil.

[0020] In one embodiment, this invention further provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby said plant exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family member for a period of time sufficient to promote boron accumulation within said plant material.

[0021] In some embodiments, such methods further comprise the step of engineering the plant to express one or more genes in an altered manner, such that altered expression of said one or more genes results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof.

[0022] In another embodiment, this invention provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby a shoot in said plant contains intracellular complexes of malic acid, fructose, glucose, citric acid or a combination thereof with boron reducing toxicity of boron to said plant, thereby being a method of increasing boron tolerance in said plant. In some embodiments, according to this aspect, such method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms said intracellular complexes.

[0023] In some embodiments, according to this aspect, the plant is engineered to overexpress phosphoenol pyruvate carboxylase, Core Binding Factor β family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.

[0024] In some embodiments, the gene is from an alternate plant species than that of said plant, or in some embodiments, the gene is bacterial in origin. In some embodiments, the expression of the gene is inducible.

[0025] In some embodiments, the invention provides a plant engineered in accordance with the methods of this invention and in some embodiments, the invention provides a seed of a plant or a transgenic plant thus engineered.

[0026] In some embodiments, the plant or seed is of a plant comprising a crop, flowering plant, grainbearing plant, fruitbearing plant, nutbearing plant, herb, turf grass, sod or seedling.

[0027] In some embodiments, the method further comprises the step of engineering the plant to under-express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof.

[0028] In another embodiment, this invention provides a method for reducing soil boron content, the method comprising:

❖ establishing growing plants in soil containing an undesirable concentration of boron, wherein the plants exhibit increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants or in a plant of a related family member; and

❖ severing and removing the plants from the soil.

[0029] According to this aspect and in one embodiment, the method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms s intracellular complexes with boron and such an engineered plant is then grown in the soil. [0030] According to this aspect and in some embodiments, the soil is irrigated with desalinated water. According to this aspect and in some embodiments, the soil bounds or lines a water reservoir.

DESCRIPTION OF THE DRAWINGS

[0031] Various embodiments of the solid supports, methods, plants and seeds of the invention are described herein with reference to the figures wherein:

[0032] Figure 1 depicts hydroponic growth of Arabidopsis and Thellungiella. Figure 1A presents a top view, plantlets growing in rockwool-filled test tubes. Figure IB depicts a root system hanging down from the 4 cm test tubes (lifted out of the aerated nutrient solution for photography and exposing the red air-stones). For measurements the roots were gently pried separate while in water.

[0033] Figure 2 shows the effect of boron on Arabidopsis and Thellungiella grown in soil. Figure 2A depicts thaliana and T. halofila plants watered for 23 days with solutions containing boric acid at the indicated concentrations. The photos were taken on day 23 after start of boron exposure. Note the relative tolerance of Thellungiella to boron. Similar effects were observed in at least five independent experiments. Figure 2B graphically depicts the concentration of accumulated boron, [B]int, in the shoot of plants harvested on day 23 of exposure as a function of boron concentration in the irrigation solution, [B]ext, expressed as mmol per kg of fresh weight, FW, of the whole shoot. Symbols: mean ±SE, of three replicates (each replicate consisted of 2-3 different plants of the experiment of A; a total of 9 Arabidopsis and 7 Thellungiella plants were used (except 2 replicates of each with a total of 5 plants at [B]ext of 5 mM). Where not seen, error bars are smaller than the symbols. B-treated plants accumulated more B than controls (*: p<0.002, **: p<0.001). Figure 2C graphically depicts the mean FWs of plant shoots of the experiment shown in Figure 2A, expressed as % of their respective controls, as a function of [B]int (shown in Figure 2B; note the break in the abscissa). The numbers near the symbols represent [B]ext (in mM). In control conditions, the absolute mean shoot FW of Arabidopsis was higher than FW of Thellungiella (1242 ±95 mg [±SE; n= 9] and 471 ±89 mg [n=7], respectively, p<0.001). Under Figure 2B treatments, the relative FW of Thellungiella didn't change, but that of Arabidopsis declined: not only did they differ at the same [B]ext, (**: p<0.001), but, notably, at the similar [B]int of about 33 mmol.kg-1 FW (marked by the vertical dashed line) Arabidopsis FW was much lower than Thellungiella FW (*: p<0.002). Other details as in Figure 2B.

[0034] Figure 3 depicts the effect of boron on Arabidopsis and Thellungiella grown in a hydroponic system and exposed for 7 days to boric acid at the indicated concentrations in the growth solution. Photos taken on day 7. Note the relative tolerance of Thellungiella to boron. Similar behavior was observed in at least five independent experiments. [0035] Figure 4 depicts the concentration of plant-accumulated boron, [B]int, as a function of boron concentration in the root-bathing solution, [B]ext. Figure 4A graphically depicts the accumulation in the root. [B]int is expressed in mM boron in the root "water" (FW-DW). Symbols are means (± SE) of 12-14 repeats from two independent experiments (when invisible, the error bars are smaller than the symbols). The B-treated plants accumulated more B than control plants (*: p<0.001). The dashed line denotes [B]int = [B]ext. Note, that in all cases, except the control, [B]int is below the corresponding bath boron concentration [B]ext (p«<0.001). [B]int of Thellungiella and Arabidopsis were not different. Figure 4B graphically depicts the accumulation in the shoot (the same experiments as in A). [B]int is expressed as mM boron in "shoot water" (FW-DW). Symbols represent means (±SE) of 7-8 replicates. All [B]int values are larger that the [B]int of the respective controls (*: p<0.001; when not seen, the error bars are smaller than the symbols). In control conditions, at [B]ext of 0.005 mM, [B]int was higher in Thellungiella than in Arabidopsis (#: p<0.001), but at [B]ext of 10 mM [B]int was higher in Arabidopsis than in Thellungiella ($: p<0.01). Figure 4C graphically depicts the dependence of shoot FW on the shoot [B]int. Symbols: Mean relative FW (±SE) of a shoot expressed as % of their respective mean control FWs (separately for each experiment), as a function of [B]int (shown in Figure 4B). The numbers near the symbols represent [B]ext (in mM) and C - the control [B]ext (0.005 mM). In control conditions, the absolute mean shoot FW of Arabidopsis was higher (p<0.01) than FW of Thellungiella (2413 ± 531 mg [+SE; n=7] and 494 ± 47 mg [n=7], respectively). Among the B-treated plants, the relative shoot FW of Thellungiella did not change, but that of Arabidopsis declined to 45 and 36 % of control at the respective exposures to 5 and 10 mM B (*: p<0.02, **: p<0.01), and was lower than Thellungiella relative FW at the same [B]ext ($: p<0.001). We note, in particular, the difference ($:p<0.001) between the FWs of the two plant species at the same [B]int of about 12 mM (indicated by the vertical dashed line). Figures 4D-4E graphically depict the amount of water transpired during a day (8 hs) by soil-grown Arabidopsis and Thellungiella. Prior to transpiration measurements plants were irrigated for 5 days (4D) or for 14 days (B) by either DDW (Control) or by DDW with 5 mM B (B (5)). Figure 4D depicts the mean transpiration (±SE) determined in three (Control) or two (B(5)) independent experiments from the indicated number of pots (each with 2-4 plants; see detailed description of measurement below). 5 days of boron treatment did not affect transpiration, in agreement with other experiments with soil grown plants, where 5 days of B treatment did not affect plant appearance. In parallel, in control conditions, Thellungiella transpired on average about 50 % more than Arabidopsis (*: p<0.02; 2-tail t test). Figure 4E depicts an independent experiment similar to Figure 4D. Here, under B-treatment, Arabidopsis transpired about 30% less than control Arabidopsis (#: p< 0.1, 2-tail t test, or, p<0.05, single-tail t test). In control conditions, the transpiration of Arabidopsis and Thellungiella did not differ (p>0.2). Other details as in Figure 4D. In summary, in none of the four independent experiments did Arabidopsis transpire more than Thellungiella. If the same relative level of transpiration was preserved during Figure 4E treatment, passive boron accumulation in Arabidopsis would not be expected to exceed that in Thellungiella. Figures 4F and 4G Determining leaf surface area for quantification of transpiration. Figure 4F shows a scan of leaves (table-top office scanner HP7130) of plants from one pot. Figure 4G shows the conversion to contours and calculation of the enclosed area as preformed by ImageJ software (by W. Rasband, NIH, USA, http://rsb.info.nih.gov/ij). In control conditions, the mean FW of a unit leaf area was similar in soil-grown Arabidopsis and Thellungiella (22.5 ±1.8 (± SE) and 21.5 ±0.2 mg.cm-2, respectively, obtained by dividing the WF of the shoot by the surface area of its leaves. These values are from 6 pots and 8 pots, respectively, each with 2-4 plants).Transpiration measurements: Daily water loss, W, was determined after the treatment period by weighing each pot with plants in the morning and 8 hours later in the evening (on a Sartorius BP4100S balance, resolution d=0.01 g). Two measurements were performed on each pot on two consecutive days, and averaged. Direct evaporation from the soil was minimized by covering the pot beneath the rosette with aluminum foil. Residual "blank evaporation", be, from a similarly covered empty pot with similarly perforated aluminum foil was subtracted from each W measurement. The leaf area, A, was determined as shown. The transpiration, T, was calculated as: T= ( W - be ) / A.

[0036] Figure 5 depicts the Boron accumulation in the shoot sap. Boron concentration in the sap, [B]SAP (in mmol B .kg-1 sap weight, SW) vs. B concentration in the whole shoot of plants, [B]int (in mmol B .kg-1 FW), in the same experiments. In addition to controls, B treatments were 0.2, 1 and 5 mM (the highest exposure is denoted by numbers near the symbols). Dotted line: [B]SAP = [B]int. The data are from two experiments: from soil (S)-grown and from hydroponics (H)-grown plants. Symbols: mean values of [B] (± SE, n=2 to 6 replicates, each consisting of 3 plants) in fresh weight of sap extracted by centrifugation. In both plant species [B]sap is similar to [B]int up to roughly [B]int= 12 mmol B .kg-1 FW. Above this concentration, for example in Arabidopsis at [B]int of 13 and 25 mmol B .kg-1 FW, [B]SAP was lower than [B]int (roughly 10 and 14 mmol B .kg-1 SW, respectively; *: p<0.05, **: p<0.01). This relationship justifies the use of FW (at low [B]int) as a basis for estimation of boron dissolved in shoot sap solution.

[0037] Figure 6 depicts the abundance of polyol metabolites in the shoots of Thellungiella and Arabidopsis grown in hydroponics. Figure 6A depicts the mean concentrations (±SE, n=4-6) of individual most prominent polyol metabolites determined in two independent experiments (Expts. Ill and IV), in plants exposed to 0.005 mM B (Control) or to 5 mM B (+5 B). The mean concentrations of the metabolites were higher in Thellungiella than in Arabidopsis under the same treatments (*: p< 0.02, **: p<0.01, ***: p< 0.001; see also Table 2, Expts. Ill, IV). Figure 6B depicts the mean ratio (±SE) obtained by dividing the summed concentrations of the major metabolites in each individual sample (see SUMa in Table 4) by [B]int from the corresponding experiment (see RATIOa, Table 4). Note the break in the ordinate. The dashed line indicates ratio = 2, the number of metabolite molecules ideally complexed with one borate molecule (see text for references). The ratio values were higher in Thellungiella than in similarly treated Arabidopsis (*: p<0.01, **: p<0.001, two-tail t tests), and they were higher than '2' in Thellungiella and lower than '2' in B-treated Arabidopsis (#: p<0.05, ##: <0.005; ###: <0.0005; single-tail t tests), indicating B complexation as a way for coping with boron excess, characteristic for Thellungiella and not for Arabidopsis. Figures 6C-E depict the abundance of metabolites in the shoots of Thellungiella & Arabidopsis. Concentrations of individual most prominent metabolites were determined by GC-MS analysis in hydroponically-grown plants in two independent experiments (a single repeat each). Shown are data from 1 mM boron-treated plants (+B, +1 B), and from control plants (-B, Control). Metabolites data from additional boron treatments in these experiments are in Table 2. Figure 6C depicts Experiment I whereas Figure 6D depicts Experiment II and Figure 6E depicts the mean ratio (RATIO) between the summed concentrations of the metabolites shown in Figure 6C and 6D (SUMa) to [B]int, in the same extract samples. The dashed line indicates RATIO = 2. The mean RATIO in B-treated Thellungiella (31) was significantly larger than the mean RATIO in Arabidopsis (0.72) (*: p<0.05, one -tailed t test); but because of the great variability of the individual values both mean RATIOs Ratios differed from '2' only at a low significance level (Is: p<0.1; single-tail t test). Note the break in the ordinate. These data appear also in Table 4.

DETAILED DESCRIPTION OF THE INVENTION

[0038] This invention provides, in some embodiments, for the increased accumulation of boron within plant materials and use of the same for boron removal from water and soil sources. Related applications of the same are described herein, as well.

[0039] In some embodiments, increased boron accumulation within plant materials is effected by providing conditions such that an increased accumulation of malic acid, fructose, glucose, citric acid or a combination thereof is effected in plant shoots of interest, which malic acid, fructose, glucose, citric acid or a combination thereof are available for complexation with boron, providing an intracellular boron detoxification process in such plants. In some embodiments, conference of boron tolerance to plant materials is a contemplated result of the same.

[0040] As exemplified herein, when 1-10 mM boron was applied to Arabidopsis grown in soil or in hydroponics, the shoot internal boron concentrations, [B]int, exceeded those applied and, among other toxicity symptoms, reduced shoot FW in Arabidopsis. When the same was applied to Thellungiella, however, unlike Arabidopsis, Thellungiella had no visible toxicity symptoms. In some embodiments, this potential may be exploited to increase boron accumulation within plant materials providing an effective boron detoxification material and applications thereof.

[0041 ] Example 4 describes the harnessing of this finding to provide materials and methods for the reduction of boron concentration in water, in a manner that is commercially meaningful.

[0042] In some embodiments, the term "increased accumulation of malic acid, fructose, glucose, citric acid or a combination thereof" refers to a plant material exhibiting increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at concentrations ranging from 2 mmol kg^"1 fresh weight (FW) to 20 mmol kg^"1 FW.

[0043] In some embodiments, the term "increased accumulation of malic acid, fructose, glucose, citric acid or a combination thereof" refers to plant material exhibiting increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration in comparison to the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in plants of related family members, etc.

[0044] In some embodiments, this invention provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water.

[0045] In some embodiments, such method and materials for use in accordance with such purpose is to reduce the boron concentration in water sources, which sources are derived from desalinated water, industrially contaminated water, or any other appropriate water source, whereby such water source contains an undesirable concentration of boron, whose reduction is desired.

[0046] In some embodiments, according to this aspect, the plant material for use in accordance with the methods and for incorporation within the materials according to this aspect is fresh or dried. In some embodiments, the plant material for use in accordance with the methods as herein described is ground, chopped or further processed.

[0047] In some embodiments, the phrase "plant material" refers inter alia, to any element of a plant which is useful in accordance with the methods as herein described. In some embodiments, the phrase "plant material" refers to whole plants, cuttings of plants, shoots, leaves, and plant extracts and combinations thereof. [0048] In some embodiments, plant material specifically includes materials derived from a Thellungiella salsuginea plant.

[0049] In some embodiments, according to this aspect, the plant material for use in accordance with the methods and for incorporation within the materials according to this aspect is derived from a Thellungiella salsuginea plant.

[0050] In some embodiments, according to this aspect, the water is contacted with a solid support comprising said plant material, and in some embodiments, such solid support includes a column or filtering device.

[0051] In some embodiments, according to this aspect, the plant material for use in accordance with the methods and for incorporation within the materials according to this aspect exhibit increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof and is grown by hydroponics within said water. In some embodiments, according to this aspect, the plant material is derived from a plant engineered to over-express one or more genes, whose over-expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms s intracellular complexes with boron.

[0052] In some embodiments, according to this aspect, the plant material is derived from a plant engineered to over-express phosphoenol pyruvate carboxylase, Core Binding Factor β family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.

[0053] In some embodiments, according to this aspect, the plant material is derived from a plant engineered to under-express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof which in turn forms intracellular complexes with said boron.

[0054] In some embodiments, the method further includes a step to increase the production of malic acid, fructose, glucose or citric acid within Thellungiella or other plant materials as herein described by growing the same in high salt conditions.

[0055] In some embodiments, the invention provides a solid support comprising a plant material exhibiting an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof. In some embodiments, the solid support comprises a plant material exhibiting an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family, etc. [0056] In some embodiments, according to this aspect, the plant material is fresh or dried.

[0057] In some embodiments, according to this aspect, the plant material is derived from a Thellungiella salsuginea plant.

[0058] In some embodiments, according to this aspect, the solid support is a column or filtering device.

[0059] In some embodiments, a column as envisioned herein may be prepared by any conventional means, as will be appreciated by the skilled artisan. In some embodiments, the column may be packed by mixing crushed plant material with sand in various proportions and packing the column with the same. In some embodiments, in addition to varying the proportion of components, the granule size may be adjusted to manipulate the column resistance to flow. In some embodiments, a porous fused silica glass serves as an exit filter.

[0060] In some embodiments, this invention provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a brown algae material for a period of time sufficient to promote boron accumulation within said brown algae material and reduction of boron concentration in said water.

[0061] In some embodiments, this invention provides a solid support comprising a brown algae material, which complexes with boron.

[0062] In some embodiments, according to these aspects, the brown algae is Saragassum vulgaris.

[0063] In one embodiment, this invention provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby said plant exhibits increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof.

[0064] In some embodiments, such methods further comprise the step of engineering the plant to express one or more genes in an altered manner, such that altered expression of said one or more genes results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof.

[0065] It is known that metabolic pathways can be biased by overexpression of certain genes, by knockout/downmodulated expression of some genes and in some cases by combinations thereof to result in an accumulation or enhanced production of a desired product of such pathway.

[0066] In another embodiment, this invention provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby a shoot in said plant contains intracellular complexes of malic acid, fructose, glucose, citric acid or a combination thereof with boron reducing toxicity of boron to said plant, thereby being a method of increasing boron tolerance in said plant.

[0067] In some embodiments, according to this aspect, such method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms said intracellular complexes.

[0068] In some embodiments, according to this aspect, the plant is engineered to overexpress phosphoenol pyruvate carboxylase, Core Binding Factor β family -3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof [.

[0069] Methods for overexpression or downmodulated/abrogated expression are well known in the art, and may be carried out, for example, as described in United States Patent Application 20060005265.

[0070] Any appropriate method for effecting such expression is envisioned.

[0071] In some embodiments, such methods make use of a vector construct comprising a nucleic acid encoding the gene of interest. In some embodiments, the method is further effected by producing a plant comprising the vector, wherein the plant exhibits enhanced boron tolerance.

[0072] Genes of interest intended for expression in plants are first assembled in expression cassettes comprising a promoter. Methods, which are well known to or developed by those skilled in the art, may be used to construct expression vectors containing such gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Exemplary techniques are widely described in the art (see e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., herein incorporated by reference).

[0073] In general, these vectors comprise a nucleic acid sequence encoding the gene of interest, operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant.

[0074] Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmental-specific promoters, and inducible promoters. Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase ("LAP," Chao et al., Plant Physiol 120: 979-992 (1999), herein incorporated by reference); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH (benzofhiadiazole-7- carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (e.g. U.S. Pat. No. 5,187,267, herein incorporated by reference); a tetracycline-inducible promoter (e.g. U.S. Pat. No. 5,057,422, herein incorporated by reference); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al, EMBO J. 4: 3047-3053 (1985), herein incorporated by reference).

[0075] The expression cassettes may further comprise any sequences required for expression of mPvNA. Such sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.

[0076] A variety of transcriptional terminators are available for use in expression of sequences using the promoters of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (see e.g., Odell et al., Nature 313:810 (1985); Rosenberg et al., Gene 56: 125 (1987); Guerineau et al, Mol. Gen. Genet. 262:141 (1991); Proudfoot, Cell 64:671 (1991); Sanfacon et al, Genes Dev. 5:141 ; Mogen et al., Plant Cell 2: 1261 (1990); Munroe et al, Gene, 91 : 151 (1990); Ballas et al, Nucleic Acids Res. 17:7891 (1989); Joshi et al., Nucleic Acid Res., 15:9627 (1987); all of which are incorporated herein by reference).

[0077] In addition, in some embodiments, constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Callis et al., Genes Develop. 1 : 1183 (1987), herein incorporated by reference). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

[0078] In some embodiments of the present invention, the construct for expression of the nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Kalderon et al, Cell 39:499 (1984); Lassner et al, Plant Molecular Biology 17:229 (1991)), a plant translational consensus sequence (Joshi, Nucleic Acids Research 15:6643 (1987)), an intron (Luehrsen and Walbot, MolGen Genet. 225:81 (1991)), and the like, operably linked to the nucleic acid sequence encoding a gene of interest.

[0079] In preparing the construct comprising the nucleic acid sequence encoding a gene of interest, various DNA fragments can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation and, as appropriate, in the desired reading frame. For example, adapters or linkers can be employed to join the DNA fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.

[0080] Numerous transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259 (1982); Bevan et al., Nature 304: 184 (1983), all of which are incorporated herein by reference), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl Acids Res. 18: 1062 (1990); Spenceret al., Theor. Appl. Genet. 79: 625 (1990), all of which are incorporated herein by reference), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann, Mol. Cell. Biol. 4:2929 (1984), incorporated herein by reference)), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J., 2: 1099 (1983), herein incorporated by reference).

[0081 ] In some preferred embodiments, the (Ti (T-DNA) plasmid) vector is adapted for use in an Agrobacterium mediated transfection process (see e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are herein incorporated by reference). In some embodiments, strains of Agrobacterium tumefaciens are C58, LBA4404, EHA101, C58ClRifR, EHA105, and the like. Examples of Agrobacterium mediated transfection in turfgrasses are provided in PCT Patents WO00/04133; WO00/11138; and U.S. patent application Pub. Nos. 20030106108 Al ; 20040010816A1 ; and U.S. Pat. No. 6,646,185; all of which are herein incorporated by reference.

[0082] Construction of recombinant Ti and Ri plasmids in general follows methods typically used with the more common vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to structural genes for antibiotic resistance as selection genes. [0083] There are two systems of recombinant Ti and Ri plasmid vector systems now in use. The first system is called the "cointegrate" system. In this system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJl shuttle vector and the non-oncogenic Ti plasmid pGV3850. The use of T- DNA as a flanking region in a construct for integration into a Ti- or Ri-plasmid has been described in EPO No. 116,718 and PCT Appln. Nos. WO 84/02913, 02919 and 02920 all of which are herein incorporated by reference). See also Herrera-Estrella, Nature 303:209-213 (1983); Fraley et al, Proc. Natl. Acad. Sci, USA 80:4803-4807 (1983); Horsch et al., Science 223:496-498 (1984); and DeBlock et al, EMBO J. 3: 1681-1689 (1984), all of which are herein incorporated by reference).

[0084] The second system is called the "binary" system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available. In other embodiments of the invention, the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium- derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (e.g U.S. Patent No., 5,501,967, herein incorporated by reference). Homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.

[0085] In yet other embodiments, nucleic acids comprising the gene of interest are utilized to construct vectors derived from plant (+) RNA viruses (e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof). Generally, the inserted polynucleotide can be expressed from these vectors as a fusion protein (e.g., coat protein fusion protein) or from its own subgenomic promoter or other promoter. Methods for the construction and use of such viruses are described in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785; all of which are incorporated herein by reference.

[0086] In some embodiments of the present invention the nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (e.g. WO 93/07278; herein incorporated by reference).

[0087] Once a nucleic acid sequence encoding a gene of interest is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.

[0088] In some embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; and PCT Patent WO 95/16783; all of which are incorporated herein by reference). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g., using biolistic or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., PNAS, 87: 8526-8530 (1990); Staub and Maliga, Plant Cell, 4: 39-45 (1992), all of which are incorporated herein by reference). The presence of cloning sites between these markers allowed creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga, EMBO J., 12:601 (1993)). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'- adenyltransferase (Svab and Maliga, PNAS, 90: 913-917 (1993)). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of the RNAs encoded by the DNA molecule.

[0089] In other embodiments, vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (e.g. Crossway, Mol. Gen. Genet, 202:179(1985)). In still other embodiments, the vector is transferred into the plant cell by using polyethylene glycol ((e.g. Krens et al., Nature, 296:72 (1982); Crossway et al., BioTechniques, 4:320 (1986)); fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (e.g. Fraley et al., Biochemistry, December 23; 19(26): 6021-6029 (1980)); protoplast transformation (EP 0 292 435); direct gene transfer (e.g. Paszkowski et al., Biotechnology 24:387-392 (1992); Potrykus et al., Mol Gen Genet. 199(2): 169-177 (1985) including direct gene transfer into protoplasts (e.g. in Arabidopsis thaliana, Damm et al., Mol Gen Genet. May;217(l):6-12 (1989); in rice Meijer et al., Plant Mol Biol May;16(5):807-820) (1991)).

[0090] In still further embodiments, the vector may also be introduced into the plant cells by electroporation (e.g. Fromm, et al., Proc. Natl. Acad. Sci. USA, September;82(17):5824-5828 (1985) and Nature February 27-March 5;319(6056):791-793 (1986); Riggs and Bates Proc. Natl. Acad. Sci. USA August;83(15):5602-5606 (1986)). In this technique, plant protoplasts are electroporated in the -presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

[0091] Examples of biolistic transformation of perennial rye grass, Kentucky bluegrass, and Bermudagrass is demonstrated in PCT Patent WO00/11138; herein incorporated by reference for salt-tolerant transgenic turfgrass and for perennial ryegrass in PCT Patent WO03/076612; and U.S. Pat. No. 5,981,842; all of which are herein incorporated by reference.

[0092] In yet other embodiments, the vector is introduced through ballistic particle acceleration using devices (e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.) (see e.g., U.S. Pat. No. 4,945,050; and McCabe et al, Biotechnology 6:923 (1988); Weissinger et al., Annual Rev. Genet. 22:421 (1988); Sanford et al., Particulate Science and Technology, 5:27 (1987) (onion); Svab et al., Proc. Natl. Acad. Sci. USA, 87:8526 (1990) (tobacco chloroplast); Christou et al, Plant Physiol, 87:671 (1988) (soybean); McCabe et al, Bio/Technology 6:923 (1988) (soybean); Klein et al, Proc. Natl. Acad. Sci. USA, 85:4305 (1988) (maize); Klein et al, Bio/Technology, 6:559 (1988) (maize); Klein et al., Plant Physiol., 91 :4404 (1988) (maize); Fromm et al, Bio/Technology, 8:833 (1990); and Gordon-Kamm et al., Plant Cell, 2:603 (1990) (maize); Koziel et al, Biotechnology, 11 : 194 (1993) (maize); Hill et al., Euphytica, 85:119 (1995) and Koziel et al., Annals of the New York Academy of Sciences 792:164 (1996); Shimamoto et al, Nature 338: 274 (1989) (rice); Christou et al, Biotechnology, 9:957 (1991) (rice); Datta et al, Bio/Technology 8:736 (1990) (rice); European Appln. EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology, 11 : 1553 (1993) (wheat); Weeks et al., Plant Physiol., 102: 1077 (1993) (wheat); Wan et al., Plant Physiol, 104: 37 (1994) (barley); Jahne et al., Theor. Appl. Genet. 89:525 (1994) (barley); Knudsen and Muller, Planta, 185:330 (1991) (barley); Umbeck et al., Bio/Technology 5:263 (1987) (cotton); Casas et al., Proc. Natl. Acad. Sci. USA, 90:11212 (1993) (sorghum); Somers et al, BioTechnology 10: 1589 (1992) (oat); Torbert et al., Plant Cell Reports, 14:635 (1995) (oat); Weeks et al., Plant Physiol., 102:1077 (1993) (wheat); Chang et al, WO 94/13822 (wheat) and Nehra et al, The Plant Journal, 5:285 (1994) (wheat); all of which are herein incorporated by reference).

[0093] In addition to direct transformation, in some embodiments, the vectors comprising a nucleic acid sequence comprising the gene of interest are transferred using Agrobacterium- mediated transformation (Hinchee et al., Biotechnology, 6:915 (1988); Ishida et al., Nature Biotechnology June; 14(6):745-50 (1996), all of which are herein incorporated by reference). Heterologous genetic sequences (e.g., nucleic acid sequences operatively linked to a promoter of the present invention) can be introduced into appropriate plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell, Science, 237: 1176 (1987)). Species, which are susceptible infection by Agrobacterium, may be transformed in vitro.

[0094] Further examples of methods for transforming ryegrasses, turfgrasses and plants of the present invention are U.S. Pat. Nos. 6,486,384; 5,981,842; 5,948,956; 6,646,185; 6,489,166; 6,646,185; U.S. patent application Pub. Nos. 20020188964A; 20030106108A1 ; 20030217386A1 ; 20030101644A1 ; 20040003434A1 ; 20040010816A1 ; 20030106108 Al ; all of which are herein incorporated by reference.

[0095] After selecting for transformed plant material that can express a gene of interest, whole plants are regenerated. Plant regeneration from cultured protoplasts is described in Evans et al, Handbook of Plant Cell Cultures, Vol. 1 : (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. Ill, 1986, herein incorporated by reference. It is known that many plants can be regenerated from cultured cells or tissues or parts, including but not limited to all major species of turfgrass, sedges, rushes, ornamental grasses, ornamental sedges, ornamental rushes, warm (hot) season grasses, cool (cold) season grasses, fodder plants, and vegetables, and monocots (e.g., the plants described above). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.

[0096] Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.

[0097] Transgenic lines can be established from transgenic plants by tissue culture propagation. The presence of nucleic acid sequences comprising the gene of interest may be transferred to related varieties by traditional plant breeding techniques.

[0098] In some embodiments, the gene is from an alternate plant species than that of said plant, or in some embodiments, the gene is bacterial in origin. In some embodiments, the expression of the gene is inducible.

[0099] In some embodiments, the method further comprises the step of engineering the plant to under-express or to fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof.

[00100] In some embodiments, such under-expression or abrogated expression may be effected by any of the many standard means known in the art. In some embodiments, reducing expression of a desired gene will utilize expression of antisense transcripts. Antisense RNA has been used to inhibit plant target genes in a tissue-specific manner (e.g., van der Krol et al. Biotechniques 6:958-976 (1988), herein incorporated by reference). Antisense inhibition has been shown using the entire cDNA sequence as well as a partial cDNA sequence (e.g., Sheehy et al. Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); Cannon et al. Plant Mol. Biol. 15:39-47 (1990), herein incorporated by reference). There is also evidence that 3' non-coding sequence fragment and 5' coding sequence fragments, containing as few as 41 base-pairs of a 1.87 kb cDNA, can play important roles in antisense inhibition (Chang et al. Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989), herein incorporated by reference).

[00101] In some embodiments, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression.

[00102] Furthermore, for antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full-length nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred.

[00103] Catalytic RNA molecules or ribozymes can also be used to inhibit expression of the target gene or genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.

[00104] A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch-viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, Solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff, et al. Nature 334:585-591 (1988). Ribozymes targeted to the mRNA of a lipid biosynthetic gene, resulting in a heritable increase of the target enzyme substrate, have also been described (Merlo A O et al, Plant Cell 10: 1603-1621 (1998), herein incorporated by reference).

[00105] Another method of reducing expression of a desired gene utilizes the phenomenon of co-suppression or gene silencing (See e.g., U.S. Pat. No. 6,063,947, herein incorporated by reference). The phenomenon of co-suppression has also been used to inhibit plant target genes in a tissue-specific manner. Co-suppression of an endogenous gene using a full-length cDNA sequence as well as a partial cDNA sequence (730 bp of a 1770 bp cDNA) are known (e.g., Napoli et al. Plant Cell 2:279-289 (1990); van der Krol et al. Plant Cell 2:291-299 (1990); Smith et al. Mol. Gen. Genetics 224:477-481 (1990), herein incorporated by reference). [00106] Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.

[00107] For co-suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that are overexpressers. A higher identity in a shorter than full-length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used.

[00108] Another method to decrease expression of a gene (either endogenous or exogenous) is via siRNAs. siRNAs can be applied to a plant and taken up by plant cells; alternatively, siRNAs can be expressed in vivo from an expression cassette. RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to target a specific gene product, resulting in post- transcriptional silencing of that gene. This phenomenon was first reported in Caenorhabditis elegans by Guo and Kemphues Cell, 81(4):611-620 (1995) and subsequently Fire et al. Nature 391 :806-811) (1998) discovered that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in the in vitro RNA preps, that is responsible for producing the interfering activity. The present invention contemplates the use of RNA interference (RNAi) to downregulate the expression of target genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof. In preferred embodiments, the dsRNA used to initiate RNAi, may be isolated from native source or produced by known means, e.g., transcribed from DNA. The promoters and vectors described in more detail below are suitable for producing dsRNA. RNA is synthesized either in vivo or in vitro. In some embodiments, endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. In other embodiments, the RNA is provided transcription from a transgene in vivo or an expression construct. In some embodiments, the RNA strands are polyadenylated; in other embodiments, the RNA strands are capable of being translated into a polypeptide by a cell's translational apparatus. In still other embodiments, the RNA is chemically or enzymatically synthesized by manual or automated reactions. In further embodiments, the RNA is synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA may be used with no or a minimum of purification to avoid losses due to sample processing. In some embodiments, the RNA is dried for storage or dissolved in an aqueous solution. In other embodiments, the solution contains buffers or salts to promote annealing, and/or stabilization of the duplex strands.

[00109] In some embodiments, the dsRNA is transcribed from the vectors as two separate stands. In other embodiments, the two strands of DNA used to form the dsRNA may belong to the same or two different duplexes in which they each form with a DNA strand of at least partially complementary sequence. When the dsRNA is thus produced, the DNA sequence to be transcribed is flanked by two promoters, one controlling the transcription of one of the strands, and the other that of the complementary strand. These two promoters may be identical or different. In some embodiments, a DNA duplex provided at each end with a promoter sequence can directly generate RNAs of defined length, and which can join in pairs to form a dsRNA. See, e.g., U.S. Pat. No. 5,795,715; incorporated herein by reference. RNA duplex formation may be initiated either inside or outside the cell.

[00110] Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA molecules containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. The length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.

[00111] There is no upper limit on the length of the dsRNA that can be used. For example, the dsRNA can range from about 21 base pairs (bp) of the gene to the full length of the gene or more. In one embodiment, the dsRNA used in the methods of the present invention is about 1000 bp in length. In another embodiment, the dsRNA is about 500 bp in length. In yet another embodiment, the dsRNA is about 22 bp in length. In some preferred embodiments, the sequences that mediate RNAi are from about 21 to about 23 nucleotides. That is, the isolated RNAs of the present invention mediate degradation of the target RNA (e.g., major sperm protein, chitin synthase, or RNA polymerase II).

[00112] The double stranded RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi for the target RNA. In one embodiment, the present invention relates to RNA molecules of varying lengths that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi cleavage of the target mRNA. In a particular embodiment, the RNA molecules of the present invention comprise a 3' hydroxyl group. In some embodiments, the amount of target RNA is reduced in the cells of the plant exposed to target specific double stranded RNA as compared to cells of the plant or a control plant that have not been exposed to target specific double stranded RNA.

[00113] In still further embodiments, knockouts may be generated by homologous recombination. In some embodiments, knockouts may be generated by heterologous recombination. In some embodiments knockouts may be generated by Agrobacterium transfer- DNA. Generally, plant cells are incubated with a strain of Agrobacterium that contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described.

[00114] Homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.

[00115] In some embodiments, the invention provides a plant engineered in accordance with the methods of this invention and in some embodiments, the invention provides a seed of a plant or a transgenic plant thus engineered. [00116] In some embodiments, the plant or seed is of a plant comprising a crop, flowering plant, grain-bearing plant, fruit-bearing plant, nut-bearing plant, herb, turf grass, sod or seedling.

[00117] In some embodiments, such plant or seed is of a plant comprising a banana plant, strawberry plant, blackberry plant, blueberry plant, peach tree, nectarine trees, pear tree, apple tree, grape vine, vegetable plant, pine tree, olive tree, oil palm tree, rubber tree, coffee plant, cotton plant, ornamental plant, flower, flowering, bulb producing plant and others, as will be appreciated by the skilled artisan.

[00118] It is to be understood that the invention relates to methods for promoting boron tolerance in a plant, and for any plant or plant product obtained in accordance with the methods of this invention. Such plants and plant products include, inter alia, crops, fruit, or agricultural product.

[00119] The term "crop" should be understood to include the plain meaning of the word, and includes cultivated plants and agricultural produce. Crops thus obtained may be cultivated for, e.g., food, medical or industrial use. Crops which may be derived from boron-tolerant plants obtained in accordance with the methods of this invention may include but are not limited to vegetables, e. g., tomatoes, peppers, corn, potatoes, celery; grains, e. g., rice, barley, wheat; nuts, e. g., almonds, pecans, peanuts; and fruit. Other examples of crops which may be derived from boron-tolerant plants obtained in accordance with the methods of this invention include but are not limited to cacao, sugar cane, sugar beets, coffee beans, rubber latex, cotton and flower bulbs.

[00120] The term "fruit" should be understood to include the plain meaning of the word, and includes an edible, usually sweet and fleshy, ovary of a seed-bearing plant or the spore-bearing structure of a plant that does not bear seeds. As such, fruits are a subclass of plant crops or products. Fruits which may be obtained in accordance with the methods of this invention include but are not limited to grapes, bananas, peaches, nectarines, pears, apples, grapefruit, tangerines, lemons, limes, and berries, such as strawberries, blackberries and blueberries.

[00121] The term "agricultural substance" should be understood to include the plain meaning of the word, and includes plants and their components, such as roots, stems foliage, flowers, etc., crops, harvested crops, and mixtures thereof. Exemplary agricultural substances include vegetable crops and plants, berry fruit crops and plants, berry fruit bushes, flowers, ornamental bushes, pome fruit trees and crops, such as apples and pears, stone fruit trees and crops, such as peaches and plums, grain crops and plants, bulbs, seeds, tubers, turf grass, fruit plants (e. g., bananas), vine crops and plants, tobacco plants, ornamental trees, commodity crops, plants and trees, medicinal plants and herbs. [00122] The present invention is not limited to any particular type of plant. Indeed a variety of plants are contemplated. In some embodiments, said plant is chosen from one or more members of a grass family, a sedge family and a rush family. In some embodiments, said plant comprises one or more of annual and perennial plants. In some embodiments, the plant is a warm season plant. In one embodiment, said warm season plant is a turfgrass. In other embodiments, said turfgrass is one or more of bahiagrass, Bermudagrass, centipedegrass, St. Augustine grass, zoysiagrass, carpetgrass, centipedegrass, buffalograss, hurricanegrass, seashore paspalum and the like. The turfgrass of the present invention is not limited to wild-type turfgrass. Indeed a variety of turfgrasses are contemplated. In some embodiments, said turfgrass is one or more of a wild- type turfgrass. In some embodiments, said turfgrass is one or more of a sport, selectively bred, and cultivator. In some embodiments, said turfgrass is one or more of a cloned plant, transgenic plant, and the like. The present invention is not limited to any particular type of ornamental grass and ornamental sedge. Indeed a variety of ornamental grasses and ornamental sedges are contemplated. In one embodiment, said ornamental grass is an Indian grass. In one embodiment, said ornamental sedge is one or more of Cyperaceae; for example Carex spp., Scirpus spp., Cyperus spp., and the like. The present invention is not limited to any particular type of rush. In one embodiment, said rush is one or more of Juncaceae; for example Juncus spp., Luzula spp., Eleocharis spp., Equisetum spp., Hierochloe spp., Hystrix spp., and the like. In some embodiments, the plant is a cold season plant. The present invention is not limited to any particular cold season plant. In one embodiment, said cold season plant is a turfgrass. In some embodiments, said turfgrass is one or more of bluegrass (e.g. Kentucky bluegrass), tall fescue, Italian ryegrass and perennial ryegrass and the like. In other embodiments, said transgenic plant is a fodder plant. In some embodiments, said fodder plant is one or more of fescues, Sudan grass, clover, alfalfa, legumes, forage grasses, bentgrass, redtop, fiorin grass (e.g. Agrostis spp.); bluegrass (e.g. Poa spp.); Columbus grass (Sorghum almum); fescue (e.g. Festuca spp.); Napier, elephant grass (Pennisetum purpureum); orchard grass (Dactylis glomerata); Rhodes grass (Chloris gayana); Sudan grass (Sorghum vulgare var. sudanense); Timothy grass (Phleum pratense), and the like. In some embodiment a legume is one or more of birdsfoot trefoil (Lotus corniculatus); lespedeza (Lespedeza spp.); kudzu (Pueraria lobata); sesbania (Sesbania spp.); sainfoin, esparcette (Onobrychis sativa); sulla (Hedysarum coronarium), and the like.

[00123] In some embodiments, said turfgrass is one or more of a wild-type turfgrass. In some embodiments, said turfgrass is one or more of a sport, selectively bred, and cultivator turfgrass. In some embodiments, said turfgrass is one or more of a cloned plant, transgenic plant, and the like. The present invention is not limited to any particular type of grass, sedge and rush. Indeed a variety of ornamental grass, ornamental sedge and ornamental rush are contemplated. In one embodiment, said ornamental grass is an Indian grass. In one embodiment, said ornamental sedge is one or more of Cyperaceae; for example Carex spp., Scirpus spp., Cyperus spp., and the like. The present invention is not limited to any particular type of rush. In one embodiment, said rush is one or more of Juncaceae; for example Juncuss spp., Luzula spp., Eleocharis spp., Equisetum spp., Hierochloe spp., Hystrix spp., and the like. The present invention is not limited to any particular type vegetative propagation. Indeed a variety of ways to provide vegetative propagation are contemplated. In other embodiments, said plant comprises one or more parts for vegetative propagation. In other embodiments, said parts for vegetative propagation comprises one or more sprigs, plugs, stolons, rhizomes, callus, meristem and sod. In other embodiments, said transgenic plant is a seed. In other embodiments, said transgenic plant is a tiller. In other embodiments said transgenic plant comprises a cold season plant. The present invention is not limited to any particular cold season plant. In one embodiment, said cold season plant is a turfgrass. In some embodiments, said turfgrass is one or more of bluegrass (e.g. Kentucky bluegrass), tall fescue, Italian ryegrass and perennial ryegrass and the like. In other embodiments, said transgenic plant is a fodder plant. In some embodiments, said fodder plant is one or more of fescues, Sudan grass, clover, alfalfa, legumes, forage grasses, bentgrass, redtop, fiorin grass (e.g. Agrostis spp.); bluegrass (e.g Poa spp.); Columbus grass (Sorghum almum); fescue (e.g. Festuca spp.); Napier, elephant grass (Pennisetum purpureum); orchard grass (Dactylis glomerata); Rhodes grass (Chloris gayana); Sudan grass (Sorghum vulgare var. sudanense); Timothy grass (e.g. Phleum pratense), and the like. In some embodiment a legume is one or more of birdsfoot trefoil (e.g. Lotus corniculatus); lespedeza (e.g. Lespedeza spp.); kudzu (e.g. Pueraria lobata); sesbania (e.g. Sesbania spp.); sainfoin, esparcette (e.g. Onobrychis sativa); sulla (e.g. Hedysarum coronarium), and the like.

[00124] As exemplified herein, there was an observable difference in boron accumulation within Thellungiella specifically, as opposed to Arabidopsis.

[00125] The difference in shoot accumulation of boron between Thellungiella and Arabidopsis is not due to passive deposition of boron carried by the transpiration stream. This is because assuming a constant transpiration rate of about 40 mg.cm-2.day-l (Fig. 4D-4E), and based on the same leaf FW per leaf unit area of 22 mg.cm-2 (there), the amount of boron accumulated during 23 days (in soil) at [B]ext of 0.005, 1, 5 or 10 mM would reach, respectively, 0.2, 8, 40, 200 or 400 mmol B .kg-1 of the shoot FW (using the following simplistic calculation: [B]int = [B]ext * transpired water volume /FW, Bowen 1972 Effect of environmental factors on water utilization and boron accumulation and translocation in sugarcane. Plant and Cell Physiology 13: 703-714). The corresponding [B]int accumulated during 7 days (in hydroponics) would be roughly 0.06, 2.5, 13, 60, 130 mmol B .kg-1 FW. Indeed, plants grown in soil and watered with distilled water or grown in hydroponics with 0.005 mM B had [B]int in the same ballpark, (Table 1), suggesting a passive accumulation of B by both plant species in these conditions.

[00126] In all other conditions and in both plant species, [B]int values were distinctly below the above predictions (Table 1), thus transpiration under boron exposure in hydroponics was much lower than assumed, and / or boron net entry (entry minus extrusion) became restricted upon exposure to higher-than-usual [B]ext.

[00127] Thellungiella was demonstrated herein to accumulate much less boron than Arabidopsis, which difference is not due to transpiration; as the transpiration of Thellungiella is not lower than that of Arabidopsis, based both on prior related observations and based on the results provided herein indicating that B-stressed Arabidopsis transpires less than the B-tolerant Thellungiella (Figure 4E).

[00128] It was demonstrated herein that both plants species extrude boron from the root ( root [B]int was lower than [B]ext (Fig. 4A). Two types of proteins in Arabidopsis have been linked to boron extrusion from the root, the anion- transporter-related AtBOR4 and the aquaporin NIP5;1 boric acid channel to B influx into root and Thellungiella, closely related to Arabidopsis, is very likely to harbor similar protein. Furthermore, BLAST analysis demonstrated a highly similar sequence in the genome of Thellungiella.

[00129] Thelungiella and Arabidopsis respective B tolerance / B sensitivity was demonstrated at the same level of internal concentration of accumulated boron, [B]int (in mmol B .kg-1 FW or in mM B in the "plant water"). Using the quantitative criterion of FW tolerance to boron in the Thellungiella shoot, as compared to Arabidopsis shoot, was shown, when both plants accumulated similar shoot [B]int, in the range of roughly 12-38 mmol B .kg-1 FW (Figs. 1, 3 and 4D-E). The contrast between the signs of B toxicity in Arabidopsis and their absence in Thellungiella at the same shoot [B]int demonstrates Thellungiella tolerance to boron at the level of the shoot.

[00130] The component of shoot-based B tolerance was not accompanied by an observed guttation from Thellungiella leaves, nor were the leaves wetted at any stage during the boron treatment period. An explanation suggesting specific boron compartmentalization in the shoot of Thellungiella does not account for the finding of similar boron distribution between the shoot sap and the whole shoot FW (Fig. 5).

[00131] The remarkable B tolerance of Thellungiella - demonstrated in particular when compared to Arabidopsis at the same internal accumulated B - correlates with the finding of several polyol metabolites in Thellungiella shoot, such as malic acid, fructose, glucose, sucrose and citric acid at a total concentration higher than in Arabidopsis, all previously shown already to bind boron. Complexation of boron occurs and serves as a means of mitigating its toxic effects on Thellungiella more effectively than in Arabidopsis.

[00132] Thellungiella is protected at the shoot level by a cellular mechanism of boron detoxification by complexing boron with, mainly, malic acid and fructose.

[00133] In another embodiment, this invention provides a method for reducing soil boron content, the method comprising:

❖ establishing growing plants in soil containing an undesirable concentration of boron, wherein the plants exhibit increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or a plant of a related family member; and

❖ severing and removing the plants from the soil.

[00134] According to this aspect and in one embodiment, the method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms s intracellular complexes with boron and such an engineered plant is then grown in the soil.

[00135] According to this aspect and in some embodiments, the soil is irrigated with desalinated water. According to this aspect and in some embodiments, the soil bounds or lines a water reservoir.

[00136] According to this aspect, and in some embodiments, in accordance with the methods and making use of the plants as herein described, the plants are grown in fields wherein the irrigation water, the soil or the ground water contains higher than desirable levels of boron. Plants are selected to tolerate the boron in the soil or water, and to take up boron while growing in the field. The plant material will incorporate boron into its tissues. Boron may then, inter alia, be removed from the planted area by harvesting and removing plant materials. In some embodiments, the plants may remain at their planted site indefinitely.

[00137] In some circumstances the harvested plant material must be handled as a waste product and disposed of in a conventional manner. Here the benefit is the relatively low energy costs of harvesting the plant material compared to recovering leachates or pumping ground water. [00138] In many instances a second benefit is obtained because the plant materials may be safely used for a variety of finished products.

[00139] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

[00140] It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the claims.

[00141] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of a conflict between the specification and an incorporated reference, the specification shall control. Where number ranges are given in this document, endpoints are included within the range. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges, optionally including or excluding either or both endpoints, in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where a percentage is recited in reference to a value that intrinsically has units that are whole numbers, any resulting fraction may be rounded to the nearest whole number.

[00142] In the claims articles such as "a,", "an" and "the" mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" or "and/or" between members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides, in various embodiments, all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g. in Markush group format or the like, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in haec verba herein. Certain claims are presented in dependent form for the sake of convenience, but Applicant reserves the right to rewrite any dependent claim in independent format to include the elements or limitations of the independent claim and any other claim(s) on which such claim depends, and such rewritten claim is to be considered equivalent in all respects to the dependent claim in whatever form it is in (either amended or unamended) prior to being rewritten in independent format.

[00143] The following examples describe certain embodiments of the invention and and should not be construed as limiting the scope of what is encompassed by the invention in any way.

EXAMPLES MATERIALS AND METHODS

B tolerance assays in soil grown plants

[00144] Plant material. Seeds of Arabidopsis thaliana (ecotype Columbia) and Thellungiella halophila (ecotype Shandong) were germinated in a soil mix (from Shaham, Givat Ada, Israel) composed of 30% Finish torf, 30 % vermiculite (size 3), 20% 'tuf (size l-3mm), 20% perlite (size 4). To achieve uniform germination, the seeds in pots underwent cold stratification in the dark under a plastic cover, at 4 °C Arabidopsis for 3 days, Thellungiella for 14 days. The plants germinated practically simultaneously, between two and four days after the end of the cold treatment, and were then grown under a short-day-regime (lOh L / 14 h D) at 350 μτηοΐ.πι- .β^"1 and at a constant temperature of 21 °C.

[00145] Boron application. At the time of treatment the plants were about one month old. Plants of each type were separated into three trays, each with 3 pots and 3 plants per pot. During the period of 23 days the plants were irrigated twice a week with different solutions: double- distilled water without or with B at the indicated concentrations. pH was 6.3 in the control solution and 5.9 in the boron solutions. The excess solution coming out of the pot was removed 2-3 hours later. The pH values of the excess solution (checked two weeks after the start of irrigation) in Arabidopsis and Thellungiella B-treatment pots were identical and varied between 7.5 and 7.8. The pH values of control pots effluent solution was always in the range of 7.7

(Thellungiella) to 8.1 (Arabidopsis).

B tolerance assays in hydroponically grown plants

[00146] The hydroponic system was established essentially as in (Smeets et al. 2008), with small changes. Light-proof plastic containers (33x24x13.5 cm) with covers were used. 35 holes were drilled in the cover and fitted with 4 cm long plastic tubes filled with 'rockwool' presoaked in nutrient medium (see Solutions below). The container was filled with the nutrient medium which, upon closing the cover, was continuous with that filling the tubes. The nutrient solution was replaced every 3 days and aerated continuously throughout the experiment using aquarium pumps and 'air stones'.

[00147] Plant material. The experiments in the hydroponic system (on both plant species) were performed during two periods, a year apart. The two sets of experiments differed in the sowing method and in the age of the plants when exposed to boron. In one set of the experiments (in Expts. I, II, V and VI [Fig. 4: the 'H-plants', Suppl. Figs. S-3 and S-4 and Suppl. Tables]), 4 seeds were placed on top of the rockwool in each of the tubes after the respective cold stratification treatments (on wet filter paper) and left to germinate under a 'plastic wrap' cover, which preserved humidity, under a short-day regime and temperature as described above. Following germination, the plastic wrap was increasingly perforated and then removed over the course of a few days and the plants were 'thinned' leaving only one plant per tube (Fig. S-l). In the other set of experiments (Expts. Ill and IV [Figs. 2, 3 and 5 and in Suppl. Tables]), the plants were sown in soil and underwent cold stratification, exactly as described above in "soil-grown plants', and two weeks after germination they were replanted into the rockwool tubes.

[00148] Boron application. In the first set of experiments (as detailed above), the plants were roughly five to seven weeks old (after germination) upon exposure to boron. However, in the second set of experiments (as detailed above), plants of both species were exactly seven weeks old (after germination) when exposed to boron.

[00149] At the start of boron treatment, the nutrient solution was exchanged for one containing boron at a control concentration, 0.005 mM (as in the growth medium), and one or more of the following (as indicated for the specific experiments): 0.2, 1, 5, 10 mM. The exposure to boron lasted 7 days (with one solution change after 3 days), at the end of which the plants were harvested and the fresh weight (FW) and, in some cases also dry weight (DW, after 3 days of drying at 70 °C) of shoot and/or root was determined. Prior to weighing and drying, the freshly excised roots were washed 3 times in ice-cold double-distilled water (DDW), and gently blotted. Analysis of plant boron content

[00150] Sample weighing. Fresh weight (FW) of plant parts (whole shoots, roots or sap) was determined by weighing plant parts immediately after their harvest. Dry weight (DW) was determined after the plant parts were dried in paper bags in an oven for three days at 70 °C.

[00151] Tissue sap extraction. Fresh plant parts were placed in a syringe cylinder, with several layers of gauze at the bottom. The syringe (without a plunger) was then immersed for a few seconds in liquid N2, then let thaw completely in a 15 mL test tube, and subsequently it was centrifuged at 3,200 g for 5 min. The gauze-filtered liquid sap was collected from the test tube, separately from the pellet which remained in the syringe.

[00152] Sample digestion and determination of B concentration. After weighing, 0.2-0.3 g of the plant material was placed in 50 mL polyethylene test tubes. 5 mL HNO₃ 65 % and 3 mL DDW (double-distilled water) were added to each test tube, and the test tubes were then incubated in a water bath at 90 °C for 2 hours, and subsequently at 70 °C for 18 hours. After the digestion, the test-tube volume was complemented with DDW to 10 or 20 mL and analyzed by ICP-AES (Inductively Coupled Plasma Atomic Emission Spectroscope (Acros from Spectro, Germany).

[00153] The final boron concentration in the plant material, Bpl (in μg/g), was obtained by Eq. 1 : Bpl = Bsmpl*Vsmpl* Dsmpl AVpl ,

where Bsmpl is boron concentration in the plant extract sample (in μg/g, obtained after calibration with standards and subtraction of blank), Vsmpl is the total sample volume (in cm3), Dsmpl is the sample density taken as lg.cm-3) and Wpl is the weight (fresh, dry, or fresh-dry, as indicated) of the plant material, in g. To obtain [B]int in mmol per kg FW, or per kg "plant water" (FW-DW), Bpl was divided by boron molar weight, 10.81g/mol.

Analysis of metabolites

[00154] Extraction: 250 mg fresh leaves harvested from Arabidosis and Thellungiella grown in hydroponics and exposed for 7 days to different boron concentrations (see above, "hydroponics"), were ground in liquid N₂. 1 ml of 10 % Ethanol in DDW containing 40 ppm of the internal standard Glucose 13C6 (Sigma- Aldrich, cat #: 310808) was added to each sample. The samples were vortexed for 5 min followed by sonication for 10 min. Finally the samples were centrifuged 20,000 g for 5 min and the clean supernatant was transferred to a clean tube.

[00155] Sample preparation: 100 of extract was dried under a dry N² stream at room temp. The metabolites were treated with silylation reagent (BSA (Ν,Ο- bis(trimethylsilyl)acetamide) +TMCS (trimethylchlorosilane) +Pyridine 5: 1 : 15), and the derived material was solubilized with cyclohexan. [00156] GC-MS analysis: The samples were analyzed using Trace GC Ultra gas chromatograph equipped with Thermo TR-5ms SQC (30mx0.25mm, 0.25μιη) capillary column coupled to Polaris Q ion trap mass selective detector (Thermo Scientific). The following GC-MS parameters were used in the analysis: carrier gas - helium; gas flow - lml/min; temperature of injector - 270 °C; injection mode - split (1 :50); initial oven temperature was 150 °C (for 1 min) then ramped at 3.57min to 190 °C (held for 4 min), then ramped at 127min to 280°C (held for 5 min); scan range was m/z 50-600.

Alternate Polyol Metabolite Extraction And Analysis Procedure

[00157] Ethanol extraction: 250 mg fresh leaves harvested from Arabidosis and Thellungiella grown in hydroponics and exposed for 7 days to different boron concentrations, were placed in 2 mL test tubes with screw-on caps and 0.5 mL ethanol 80 % was added. The test tubes were heated to 100 °C for 2 minutes, and the extracts were transferred to corresponding new test tubes. The extraction procedure and extract collection were repeated on the plant material twice more, with heating at a lower temperature of 80 °C. The extracts were dried at 40 °C in a chemical hood under a stream of warm air, and 800 mL of filter-sterilized DDW was added to each test tube.

[00158] The test tubes with extract were vortexed, and then centrifuged at about 15,000 g at room temperature. The liquid was decanted and filtered through a 0.45 mm Whatman cellulose acetate syringe filter (CAT#10462100). Subsequently hydrophobic materials were removed from the filtered extract by the addition of 800 mL chloroform, 2 minutes of mixing by vortex, centrifugation (as above) for 10 minutes and decantation of the top water phase with the sugars, without disturbing the lower phase.

[00159] Sample preparation: 50 mL of the extract were dried under a dry N2 stream at 60 °C. The extract was treated with silylation reagent (BSA [N,0-bis(trimethylsilyl)acetamide] +TMCS [trimethylchlorosilane]+Pyridine 5:1 : 15), and the derived material was solubilized with cyclohexan. A solution of 1 mg B .g-1 FW of diphenyl ether served as a blank standard for polyol quantitation.

[00160] Metabolite analysis in GC-MS: The samples were analyzed in a GCMS (Gas Chromatograph combined with a Mass Spectrometer, models 7890A and 5975C, respectively, from Agilent Technologies, CA). The identification of metabolites was based on injected standard solutions according to the manufacturer's specifications. Solutions

[00161] The hydroponic nutrient solution (based on Smeets et al. 2008, with a small alteration) contained: 0.505 mM KN0₃, 0.15 mM Ca(N0₃)*4H₂0, 0.1 mM NH₄H₂P0₄ , 0.1 mM MgS0₄ , 4.63 μΜ H₃B0₃, 2 μΜ EDFS (CioHi₂N₂NaFe0₈), 0.91 μΜ MnCl₂*4H₂0, 0.16 μΜ ZnS0₄*7H20 , 0.06μΜ Na₂Mo0₄*2H20, 0.03μΜ CuS0₄, all dissolved in DDW.

Statistics

[00162] Unless otherwise indicated, the significance tests were unpaired 2-tail t tests.

EXAMPLE 1:

Boron Toxicity And Boron Tolerance In Soil-Grown Arabidopsis And Thellungiella

[00163] Two model plants, Arabidopsis and Thellungiella were evaluated for toxicity effects of boron exposure. Arabidopsis plants grown in soil under a short-day regime exhibited symptoms of toxicity, mainly yellowing of leaf edges, already after a week of watering with 5 mM B, or after three days of watering with 10 mM B. In contrast, no signs of yellowing were visible in Thellungiella during this time. Transpiration in both plants species remained unaffected after 5 days of watering with 5 mM B; after 14 days it appeared diminished in Arabidopsis (by about 30 %, Fig. 1). Figure 1A demonstrates the plantlets growing in rockwool- filled test tubes and Figure IB demonstrates the Root system hanging down from the 4 cm test tubes (lifted out of the aerated nutrient solution for photography and exposing the red air-stones). The roots were gently pried separate while in water, for obtaining the measurements described. Note the relative tolerance of Thellungiella to boron. Similar effects were observed in at least five independent experiments.

[00164] Following 23 days of watering with 5 mM B, all Arabidopsis leaves were severely yellowed and necrotic at the edges and in 10 mM B Arabidopsis plants were dead (Fig . 2A). In contrast, only the older leaves of Thellungiella yellowed slightly at the edges, even after 23 days of watering with either 5 mM or 10 mM boron, an appearance not much different from the leaves of control plants and very likely due to plant ageing (Fig. 2A). This healthier appearance of Thellungiella could be attributed merely to a much lower boron concentration in the Thellungiella leaves, as compared to Arabidopsis (Figs. 2B, 2C). Indeed, while in control conditions Thellungiella contained as much B in the shoot as Arabidopsis (either 0.14 and 0.17 mmol B .kg-1 FW, respectively, in one experiment, or 1.2 mmol B .kg-1 FW in both species in another, Table 1 ), upon an exposure to boron the internal boron concentration, [B]int, increased relatively much more in Arabidopsis shoot than in Thellungiella' s. Thus, at an external boron concentration, [B]ext, of 5 and 10 mM, Arabidopsis accumulated between about 2 to over 10 times more boron than Thellungiella (Fig. 2B and Table 1 below). YISSUM7270PC 36

Table 1 :

[B]int (mmol B.kg-1 FW, or , [*]: mM in 'shoot water' )

YISSUM7270PC 37

[00165] To circumvent a possible effect of this difference in B accumulation, we compared the shoot fresh weight (FW) - a quantitative measure of plant growth - at a similar internal boron concentration. Since in the control conditions the FW of Arabidopsis was 2.6 higher than that of Thellungiella (Fig. 2B), we compared the FWs normalized to the corresponding mean control values. In the range of 5 to 10 mM of added boron, [B]ext, the shoot FW of Thellungiella did not change from its value in control conditions, while Arabidopsis relative FW dropped down dramatically, and at ([B]int) of about 33 mmol.kg-1 FW (attained in Arabidopsis irrigated with [B]ext = 5 mM and in Thellungiella - with [B]ext =10 mM, Arabidopsis FW was less than 50% of its own control while Thellungiella' s FW was not affected (Fig. 2C), suggesting that the Thellungiella shoot tolerates boron much better, employing, perhaps, a detoxification mechanism.

[00166] C: Mean FWs of plant shoots of the experiment shown in A, expressed as % of their respective controls, as a function of [B]int (shown in B; note the break in the abscissa). The numbers near the symbols represent [B]ext (in mM). In control conditions, the absolute mean shoot FW of Arabidopsis was higher than FW of Thellungiella (1242 ±95 mg [±SE; n= 9] and 471 ±89 mg [n=7], respectively, p<0.001). Under B treatments, the relative FW of Thellungiella didn't change, but that of Arabidopsis declined: not only did they differ at the same [B]ext, (**: p<0.001), but, notably, at the similar [B]int of about 33 mmol.kg-1 FW (marked by the vertical dashed line) Arabidopsis FW was much lower than Thellungiella FW (*: p<0.002). Other details as in B.

Example 2

Boron Toxicity And Boron Tolerance In Arabidopsis And Thellungiella Grown By

Hydroponics

[00167] In assays with plants grown hydroponically (which allowed for better control of B treatments and, additionally, observations of B effects on the roots), B toxicity symptoms appeared much faster than in soil grown plants. A single week of boron exposure sufficed to produce toxicity symptoms in Arabidopsis leaves resembling those observed after 3 weeks in soil-grown plants (Fig . 3).

[00168] In hydroponics, Arabidopsis showed signs of stress (yellowing of leaf edges) already at 1 mM external boron (data not shown). At an external exposure to 5 mM B, Arabidopsis growth - mainly of the shoot - was markedly impeded relative to the control and at 10 mM boron Arabidopsis plants became severely chlorotic and stunted, and the root was markedly thinner and shorter than at 5 mM B (Fig. 3). In contrast, even at 10 mM external B, YISSUM7270PC 38

Thellungiella plants appeared not much different from plants grown in control conditions (Fig. 3).

[00169] The roots of both plant species accumulated B increasingly when exposed to applied boron concentrations, [B]ext, of 0.005 to 10 mM. However, the accumulated boron concentration in both plants, [B]int, was significantly lower than B concentration in the medium, suggesting active extrusion of boron from roots. There was no difference between the two species in root [B]int (Fig. 4A). Thus it appears that the mechanism of Thellungiella tolerance does not reside entirely in the root.

[00170] Generally, the shoot concentrations of the accumulated boron, [B]int, markedly exceeded the concentrations of boron in the hydroponic solutions presented to the roots, [B]ext (Fig. 4B, Fig. 4D and Table 1), both under boron treatments and in control conditions. Interestingly, in control conditions in these experiments, Thellungiella attained, on average, 1.7 times higher shoot [B]int than Arabidopsis (0.407 ±0.027 mM, n=7, vs. 0.246 ± 0.007 mM, n= 8; Fig. 4B), and this pattern was consistent among different experiments (the mean ratio between control [B]int in Thellungiella and Arabidopsis, averaged over 6 experiments, was 2.07+0.21 (Table 1), reflecting, perhaps a relatively higher transpiration rate.

[00171] In contrast to this, and resembling soil-grown plants, upon exposure to increasing boron concentrations [B]int increased relatively more in Arabidopsis than in Thellungiella. At [B]ext of 10 mM, Arabidopsis accumulated consistently about twice as much boron as Thellungiella (45.3 ±4.7 mM, n=8, vs. 25.0 ±1.7 mM, n=7; Fig. 4B; see also Fig. 4D and Table 1). In other experiments, [B]int in Arabidopis exceeded that in Thellungiella also at [B]ext of 5 mM (e.g., Fig. 4D and Table 1). Moreover, when the two plant species were compared at the same level of accumulated internal boron, [B]int, of about 12 mM (in "shoot water"; Fig. 4C), or at about 38 mM (in "shoot water"; Fig. 4E), the relative shoot FW of Thellungiella was markedly larger than that of Arabidopsis (and also not different from the FW of control Thellungiella), resembling soil-grown plants (Fig. 1C). Thus, irrespective of their growth conditions, soil or hydroponics, at the same boron load in the shoot Thellungiella displayed consistently greater boron tolerance as compared to Arabidopsis, suggesting a shoot-based mechanism of B -tolerance in Thellungiella.

[00172] To resolve whether tolerance in Thellungiella is due to increased boron adsorption to or deposition in the cell wall (in which B is a structural component, see Introduction), we compared boron partitioning between the cell sap and the cell wall, by comparing boron concentration in the cell sap ([B]sap) with [B]int in the FW of the whole shoot. [B]sap was similar to [B]int up to about [B]int =12 mmol B .kg-1 FW in both plant species (in plants YISSUM7270PC 39

grown both in soil and hydroponically; Fig. 5 ). This similarity indicates that in Thellungiella exposed to [B]ext of 5 mM (both in soil or hydroponics, the same conditions which cause toxicity symptoms in Arabidopsis but not in Thellungiella) B is not compartmentalized preferentially in the cell sap nor is it sequestered in the insoluble tissue fraction (cell wall or intracellular insoluble particles). Therefore, compartmentalization in the cell wall is unlikely to be part of boron detoxification mechanism in the Thellungiella shoot. Only at [B]int values exceeding 13 mmol B .kg-1 FW, attained in Arabidopsis exposed to 5 mM B, did [B]sap become significantly smaller than [B]int (Fig. 5), suggesting boron enrichment in the insoluble fraction of the Arabidopsis shoot; this clearly did not impart tolerance to B and was probably due to the net loss of water from the stressed Arabidopsis plant.

Example 3

Boron tolerance and polyol metabolites

[00173] To ascertain whether the key to the greater boron tolerance of Thellungiella is a consequence of intracellular boron detoxification by polyol metabolites, the profile of several such compounds was compared in both plant species. Metabolites were extracted from plants grown hydroponically with different concentrations of boron and analyzed using GC-MS as described. A few metabolites were found in Thellungiella, such as malic acid, fructose and glucose, and, occasionally, also sucrose, both in control conditions (0.005 mM B) and in B- treated plants (Fig. 6A , and Figures 6C-6E and Table 2 ).

Table 2:: Major polyol metabolite concentration in leaf tissue extracts of Arabidopsis (Ara) and Thellungiella (The) exposed to different boron treatments, [B]ext. A: Metabolite concentrations (determined using GC-MS, as described) are single determinations. For the recited experiment numbers III-IV listed in Table 2, the metabolite concentrations were determined using GC-MS as described, and expressed as mM in the "plant water", i.e., the difference between the shoot fresh and dry weights, FW-DW. Values presented represent the mean values obtained for 2-3 repeats of each experiment. ND: 'not detected'. Polyol concentrations are consistently higher in Thellungiella than in the corresponding Arabidopsis plants, as shown in the ratio column (The/Ara). The underlined values of The/Ara were calculated assuming a value of 0.01 mmol.kg-1 FW of shoot material instead of 'ND' (to avoid division by null). Additional metabolites determined in Expts III and IV are tabulated in Table 3. B: Mean concentrations and ratios (and SE) calculated separately for each pair of the experiments. The The/Ara ratios are averages of the values in part A of the Table. In Expts. Ill and IV the mean concentrations were averaged over 4-6 pooled samples and they are presented in Figure 6A. Further analyses of these data are presented in Table 4. YISSUM7270PC 40

YISSUM7270PC 41

[00174] In a more detailed analysis in two of the four experiments (Expts III and IV), significant amounts (3-4 mM) of citric acid (Table 2) and small amounts (0.5-1.5 mM) of a few other metabolites: alanine, proline, glutamic acid and pyruvic acid (Table 3) were found. Curiously, under treatment with 5 mM B, there was more pyruvic acid (a potential boron- binding metabolite, Jones 2008) in Arabidopsis, than in Thellungiella (p<0.05; Suppl. Table 3).

[00175] The major metabolites, malic acid, fructose, glucose and citric acid (where determined) were generally more abundant in Thellungiella than in Arabidopsis, both in control conditions and under boron treatments (Fig. 6A , 6C and 6D, and Table 2), albeit, with one single exception in Expt. I in control conditions; there the glucose concentration was higher in Arabidopsis.

[00176] Since one borate molecule can ideally bind two of any of these molecules the combined concentration of these polyols were assessed for their ability to remove the internal boron from interactions within the intracellular milieu. The concentrations of malic acid, fructose, glucose and sucrose, and (in experiments III and IV) also citric acid (SUMa in the Suppl. Table 1, V) were summed, and then the ratio of this sum to [B]int was calculated in the same or corresponding shoot samples (RATIOa in Table 4). In experiments III and IV, the mean SUMa in Thellungiella exceeded SUMa in Arabidopsis by roughly three to four fold (Table 4) and the mean RATIOa was larger than '2' in Thellungiella and smaller than '2' in Arabidopsis (Fig. 6B and Table 4). This is consistent with boron removal by complexation with the polyols conferring B tolerance on Thellungiella.

[00177] If we consider only malic acid and fructose, which bind boron with much higher stability constants than glucose and sucrose, their mean combined concentrations (M+F, SUMb) in experiments III and IV were several-fold higher in Thellungiella than SUMb in Arabidopsis, under all conditions, and qualitatively, experiments I and II were consistent with these results (Table 4). Unlike Ratioa, the mean RATIOb (the ratio between SUMb and the accumulated [B]int) was significantly higher than '2' only under control conditions (Table 4), suggesting that while Thellungiella abounds in B-binding polyol metabolites relative to Arabidopsis, malic acid and fructose alone would not suffice for neutralizing the accumulated boron completely in 2: 1 polyohB complexes.

Table 3: Concentrations of metabolites in leaf tissue extracts in Experiments III and IV (complementing Table 2). Mean concentrations (± SE; mmol.kg-1 FW) of metabolites determined in 4-6 repeats pooled from both experiments. Marked in bold are values above an arbitrary cutoff of 0.5 mmol.kg-1 FW. YISSUM7270PC 42

[Metabolites] (mM, EXPTs IIIJV)

Arabidopsis Thellungiella Arabidopsis Thellungiella

Metabolites control control 5 mM B 5 mM B

Lactic_Acid AVG 0.154 0.173 0.359 0.168

SE 0.049 0.041 0.063 0.043

Pyruvic Acid AVG 0.568 0.491 1.25 0.483

SE 0.144 0.072 0.10 0.074

Alanine AVG 0.421 0.522 1.22 0.930

SE 0.040 0.150 0.15 0.132

Oxalic_Acid AVG 0.201 0.212 0.186 0.212

SE 0.023 0.016 0.025 0.023

Valine AVG 0.086 0.146 0.368 0.156

SE 0.016 0.020 0.052 0.034

Leucine AVG 0.065 0.086 0.233 0.119

SE 0.010 0.014 0.028 0.029

Isoleucine AVG 0.045 0.070 0.205 0.080

SE 0.008 0.010 0.032 0.015

Proline AVG 0.192 0.391 0.177 1.16

SE 0.041 0.184 0.028 0.25

Glycine AVG 0.038 0.067 0.102 0.068

SE 0.004 0.016 0.006 0.012

Succinic_Acid AVG 0.184 0.220 0.1501 0.170

SE 0.027 0.025 0.009 0.023

Threonine AVG 0.161 0.304 0.404 0.182

SE 0.024 0.086 0.067 0.054

Aspartic_Acid AVG 0.132 0.809 0.180 0.611

SE 0.0187 0.135 0.026 0.236

Hydroxy- Proline AVG 0.108 0.185 0.234 0.177

SE 0.012 0.044 0.013 0.022

Glutamic_Acid AVG 0.087 1.05 0.179 0.880

SE 0.018 0.31 0.036 0.333 YISSUM7270PC 43

Table 4: The combined concentration of the major accumulated polyol metabolites, SUM, and its RATIO to the concentration of boron accumulated in the same experiment, [B]int:

YISSUM7270PC 44

Table 4A:

UMa s t e com ne concentrat on o ma c ac , ructose, g ucose, sucrose an c tr c ac n a g ven samp e c tr c ac was quant ed only in experiments III and IV). SUMb is the combined concentration only of malic acid and fructose (M+F), which bind boron with higher stability constants than the other polyols (see text for references). RATIOa and RATIOb correspond to the respective SUMs divided by [B]int determined in each individual experiment. In experiments I and II, all SUM and RATIO values were calculated from single determinations. In experiments III and IV, all SUM and RATIO values are means of 2-3 samples and [B]int values are means of 3-4 samples. RATIO values exceeding the value of '2' are marked in bold. The mean concentrations of accumulated B, [B]int, were copied here for convenience from Table 1.

Qualitative conclusions from the data in the individual experiments are as follows: the values of SUMa and SUMb in Thellungiella consistently exceeded those of Arabidopsis. Further, RATIOa in Thellungiella consistently exceeded '2', both in control and at [B]ext of 1 and 5 mM.

YISSUM7270PC 45

However, Ratiob was not similarly consistent in B-treated Thellungiella: although larger than '2' in Expt. I, Ratiob did not exceed '2' in 5 mM in Expts. Ill and IV. In contrast, in B-treated Arabidopsis both RATIOa and Ratiob were always smaller than '2'.

Table 4B:

Mean SUMs and RATIOs (and SE) calculated separately for each pair of the experiments in A (in Expts. I and II n=2, in Expts. Ill and IV n=4 to 6). [B]int values are from Suppl. Table S-I. RATIO values exceeding the value of '2' are marked in bold. Superscripts indicate the significance level of the various comparisons detailed below. In Thellungiella, in Expts. Ill and IV, the mean values of SUMa and SUMb were greater than in Arabidopsis (f: p<0.001, g: p<0.01, two-tailed t test). Expts I and II were qualitatively consistent with these results. The mean values of RATIOa in Expts. Ill and IV exceeded '2' in Thellungiella and were smaller than '2' in Arabidopsis (a: p<0.0005; b: p<0.005, c: p<0.05; shown also in Fig. 6B). RATIOa of Expts. I and II agreed qualitatively with these results (shown also in Fig. 6C), suggesting together an abundance of polyol metabolites in Thellungiella (but not in Arabidopsis) roughly adequate to bind all accumulated boron at a 2: 1 ratio and thereby alleviate boron toxicity. In summary, RATIOb in Expts. I and II was qualitatively consistent with RATIOa. However, in Expts III and IV, values of RATIOb, were smaller than '2' (p<0.5) in 5 mM B-treated Thellungiella, as in Arabidopsis (a: p<0.0005), suggesting that malic acid and fructose, by themselves, could not suffice to bind all accumulated boron at the 2:1 ratio, and thus, by themselves, they could only partially alleviate boron toxicity in Thellungiella.

YISSUM7270PC 46

Example 4

Large Scale Boron Removal From Water Sources

[00178] To investigate whether plant materials as herein described can be utilized to facilitate boron removal from water sources, Boron removal via adsorption onto a fixed matrix is assessed. Raw plant material and raw dried plant material is assessed for the ability to adsorb boron. As a first evaluation, Thellungiella is assessed in this context, as it has been shown to be a superior source for boron complexation in the Examples hereinabove.

[00179] As the metabolites noted hereinabove are produced in Thellungiella naturally and without induction (i.e., without a requirement for prior boron exposure), the use of the plant, for example, in the form of dried leaves, as a filtering substance within filtering columns for boron removal from water is assessed.

[00180] Some of the envisioned methods for developing the columns for use in accordance with the large scale boron removal as herein described include filling columns with crushed filtering material (from either saline-pretreated or from non-pretreated plants), at various packing densities (manipulated by mixing the organic material in different proportions with inert silica sand) at an optimum pH), at various (pump-controlled) flow rates, and determining boron concentration in the column material and in the effluent solution, which can be collected using a fraction collector. The column is then flushed with acidified solution [brackish water acidified with HC1 to pH 5] and the boron concentration in the effluent solution is determined, as well. Such an evaluation provides a measure of a "regenerated" column in terms of recovery of filtering functionality.

[00181] Some of the methods for developing the columns for use in accordance with the large scale boron removal as herein described include soaking crushed dry leaves of the plants/plant materials as herein described (packed in inert fine-mesh net) in 6 mg/L boron solution (sea-water-like) for various durations, for example, 10, 20 and 60 minutes, on a shaker, at various values of pH: for example, 8, 8.5 both close to that of sea water, and 9.5 (one unit above the pH of sea water, and roughly similar to entry pH in the 2nd stage of sea water desalination), and additionally in 0.6 mg/L boron solution (brackish- water-like), for durations of 20, 60 and 180 minutes at pH between 7.5 (close to the pH of brackish water) and pH 9.5 and determining the values of boron and polyols in the plant material and in the solution using ICP and GCMS, respectively. These experiments may be repeated any number of times to obtain statistically reliable data.

[00182] Complexation of boron with polyols is pH dependent and boron removal from water increases with increasing pH. Acidification reverses the complexation (e.g., 15 min YISSUM7270PC 47

eliminated boron from one mg of boron-polysaccharide complex dissolved in 10 ml of water with 1 M HC1, Matoh et al., 1993), underscoring the rationale for the different pH tests noted hereinabove. The skilled artisan will appreciate that routine experimentation will result in potentially modifying any of the parameters noted hereinabove in order to optimize the methods and materials thus evaluated.

[00183] Some of methods for developing the columns for use in accordance with the large scale boron removal as herein described include growing Thellungiella for 6-8 weeks for leaf harvest as described in Lamdan et al., 2012 (Plant, Cell & Environment 35:735-746) (and additionally, for about 3 months, to renew seed stocks). In some instances, a dedicated growth chamber is used. Dry weight (DW) is determined after drying in paper bags in an oven for three days at 70 °C. (Fresh weight (FW) of plant leaves will be determined by weighing immediately after their harvest).

[00184] Thellungiella grown on non-saline water is conducted to assess whether growth in high salinity increases Thellungiella ability to adsorb /to complex B. Saturation plots of material from salt-treated versus non-treated Thellungiella are comparatively assessed.

[00185] The procedures are repeated, at the best-performing solution (the optimum pH, at the optimum duration, and also at twice the duration and at half the duration), using dried leaves from plants irrigated during their growth period with one of 3 levels of salinity: sea water, sea-water diluted 2x and 5x..

[00186] Another large scale boron removal method involves the use of brown algae, such as Saragassum vulgaris, containing a cell-wall-associated polymeric polyol, alginate, known to complex with boron. Use of raw dried Saragassum vulgaris algae will be similarly evaluated for the ability to prepare solid supports such as columns for the large scale removal of boron from water sources

[00187] S. vulgaris is collected, for example, from appropriate sources rocky and the initial boron content (in the dried and crushed algal material) is determined.

[00188] In some embodiments, the dried and crushed algal material is prewashed, to remove the initial boron prior to boron adsorption assays by soaking in (a) mildly- acidified sea water (pH 5 or pH 6, since complexation of boron with polyols decreases with decreasing pH, and (b) mildly-acidified brackish water (also at pH 5 or pH 6; and boron removal is assessed.

[00189] Sea water or brackish water intended for boron removal is alkalinized to pH 8.5 or 9.5.

[00190] Product quality validation is assessed for all samples thus obtained, according to drinking water standards.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants (for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water.

2. The method of claim 1, wherein said plant material exhibits shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof include concentrations ranging from 2 mmol kg^"1 fresh weight (FW) to 20 mmol kg^"1 FW.

3. The method of claim 1, wherein said plant material is fresh or dried.

4. The method of claim 1, wherein said plant material is derived from a Thellungiella. salsuginea plant.

5. The method of claim 1, wherein said water is contacted with a solid support comprising said plant material.

6. The method of claim 5, wherein said solid support includes a column or filtering device.

7. The method of claim 5, further comprising the step of decreasing a pH of a solvent contacted with said solid support and eluting boron from the same, thereby regenerating boron absorption capacity in said solid support.

8. The method of claim 1, wherein a plant exhibiting increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof is grown by hydroponics within said water.

9. The method of claim 1, further comprising the step of increasing pH of said water when contacted with said plant material.

10. The method of claim 1, wherein said water is desalinated water

11. The method of claim 1, further comprising the step of engineering said plant to over- express one or more genes, whose over-expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said plant material or a combination thereof, which in turn forms complexes with boron.

12. The method of claim 11, wherein said plant is engineered to over-express phosphoenol pyruvate carboxylase, Core Binding Factor β family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.

13. The method of claim 11, further comprising the step of engineering said plant to under- express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof which in turn forms intracellular complexes with said boron.

14. A solid support comprising a plant material exhibiting an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, for a period of time sufficient to promote boron accumulation within said plant material.

15. The solid support of claim 14, wherein said plant material is fresh or dried.

16. The solid support of claim 15, wherein said plant material is derived from a Thellungiella salsuginea plant.

17. The solid support of claim 14, wherein said solid support is a column or filtering device.

18. A method for reducing the boron concentration in soil, said method comprising contacting soil containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said soil.

19. The method of claim 18, wherein said contacting includes growing a plant exhibiting said increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof in said soil.

20. The method of claim 18, wherein said plant material is derived from a Thellungiella salsuginea plant.

21. The method of claim 18, further comprising the step of engineering said plant to over- express one or more genes, whose over-expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof within said plant material, which in turn forms s intracellular complexes with boron.

22. The method of claim 21, wherein said plant is engineered to over-express phosphoenol pyruvate carboxylase, Core Binding Factor β family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.

23. The method of claim 18, further comprising the step of engineering said plant to under- express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof which in turn forms intracellular complexes with said boron.

24. The method of claim 18, wherein said soil is irrigated with desalinated water.

25. The method of claim 18, wherein said soil bounds or lines a water reservoir.

26. A method for increasing boron tolerance in a plant said method comprising providing conditions whereby said plant exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants for a period of time sufficient to promote boron accumulation within said plant material.

27. The method of claim 26, further comprising the step of engineering said plant to over- express one or more genes, whose over-expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof.

28. The method of claim 27, wherein said plant is engineered to overexpress phosphoenol pyruvate carboxylase, Core Binding Factor β family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.

29. The method of claim 27, wherein said gene is from an alternate plant species than that of said plant.

30. The method of claim 27, wherein said gene is bacterial in origin.

31. The method of claim 27, wherein expression of said gene is inducible.

32. A seed produced by the method of claim 26.

33. The seed of claim 32, wherein said seed is a seed of a plant comprising a crop, flowering plant, grainbearing plant, fruitbearing plant, nutbearing plant, herb, turf grass, sod or seedling.

34. A transgenic plant produced by the method of claim 27.

35. The transgenic plant of claim 34, wherein said transgenic plant is of a plant comprising a crop, flowering plant, grain-bearing plant, fruit-bearing plant, nut-bearing plant, herb, turf grass, sod or seedling.

36. The transgenic plant of claim 34, wherein said transgenic plant is further engineered to under-express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof.

37. A method for increasing boron tolerance in a plant said method comprising providing conditions whereby a shoot in said plant contains intracellular complexes of malic acid, fructose, glucose, citric acid or a combination thereof with boron reducing toxicity of boron to said plant, thereby being a method of increasing boron tolerance in said plant.

38. The method of claim 37, further comprising the step of engineering said plant to over- express one or more genes, whose over-expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms said intracellular complexes.

39. The method of claim 38, wherein said plant is engineered to overexpress phosphoenol pyruvate carboxylase, Core Binding Factor β family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.

40. The method of claim 38, wherein said gene is from an alternate plant species than that of said plant.

41. The method of claim 38, wherein said gene is bacterial in origin.

42. The method of claim 38, wherein expression of said gene is inducible.

43. A seed produced by the method of claim 38.

44. The seed of claim 43, wherein said seed is a seed of a plant comprising a crop, flowering plant, grain-bearing plant, fruit-bearing plant, nut-bearing plant, herb, turf grass, sod or seedling.

45. A transgenic plant produced by the method of claim 38.

46. The transgenic plant of claim 45, wherein said transgenic plant is of a plant comprising a crop, flowering plant, grainbearing plant, fruitbearing plant, nutbearing plant, herb, turf grass, sod or seedling.