PHOSPHORUS USE EFFICIENCY ENHANCERS AS PLANT GROWTH PROMOTORS

Abstract

The use of a compound of the general formula (I) with the following definitions: R.sup.1 hydrogen, OH, NH.sub.2, C.sub.1-30-hydrocarbon residue which can contain one to three halogen atoms and/or one to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, R.sup.2, R.sup.3 independently hydrogen, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, it also being possible for R.sup.2 and R.sup.3 to be covalently linked to form a 5- to 8-membered heterocyclic ring, which can contain 1 or 2 halogen atoms and/or one to three further heteroatoms in addition to the heteroatoms N and O already part of the heterocyclic ring, R.sub.4 independently hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, n 0; 1; 2 or 3, 86 as plant growth promotor.

##STR00001##

Claims

1. A method for promoting plant growth comprising utilizing a compound of the general formula (I) ##STR00024## with the following definitions: R.sup.1 hydrogen, NH.sub.2, OH, C.sub.1-30-hydrocarbon residue which can contain one to three halogen atoms and/or one to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, preferably R1 is hydrogen, NH2 or a C1 to C4 hydrocarbon or C1 to C4 carboxy or carbonyl group, even more preferably, R1 is NH2, R.sup.2, R.sup.3 independently hydrogen, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, it also being possible for R.sup.2 and R.sup.3 to be covalently linked to form a 5- to 8-membered heterocyclic ring, which can contain 1 or 2 halogen atoms and/or one to three further heteroatoms in addition to the heteroatoms N and O already part of the heterocyclic ring, preferably R2 is hydrogen or a C1-C4 hydrocarbon, even more preferably R2 is hydrogen or a C1-C2 hydrocarbon, preferably R3 is a C1-C4 hydrocarbon, and even more preferably, R3 is CH3, R.sup.4 independently hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, preferably R4 is hydrogen or CH3, n 0; 1; 2 or 3, as plant growth promotor, wherein the plant growth promotor is used for biomass and/or crop yield increase, for mitigating and/or reducing the phosphate starvation stress of plants or for improving, the phosphorus or phosphate use efficiency of plants or the phosphorus or phosphate uptake of plants, the root hair development, root system architecture, drought resistance development, nutrient uptake efficiency of plants, the phosphate availability for plants, water or nutrient uptake of plants, beneficial symbiotic interactions of plants, soil microbiome or phosphorous or phosphate availability from phosphate rock or organic phosphate.

2. The method as claimed in claim 1, wherein the compound of the general formula (I) has the general formula (Ia) ##STR00025## with the following definitions: R.sup.5 hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, preferably R5 is hydrogen or CH3.

3. The method as claimed in claim 1, wherein the compound of the general formula (I) has the formula 8.14 or 8.14.6 ##STR00026##

4. The method as claimed in claim 1, wherein the compound of general formula (I) has the general formula (Ib) ##STR00027## with the following definitions: R.sup.5 hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue, which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, R.sup.6 hydrogen, C.sub.1-12-hydrocarbon residue, which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur.

5. The method as claimed in claim 1, wherein the compound of the general formula (I) has the general formula (Ic) ##STR00028## with the following definitions: R.sup.7, R.sup.8 independently C.sub.1-8-alkyl, C.sub.3-8-cycloalkyl, which can both contain one or two halogen atoms and/or one or two heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, or phenyl.

6. The method as claimed in claim 1, wherein the compound of the general formula (I) has the general formula (Id) ##STR00029## with the following definitions: R.sup.5 hydrogen, halogen, NO.sub.2.Math.C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur. R.sup.T, R.sup.8 independently C.sub.1-8-alkyl, C.sub.3-8-cycloalkyl, which can both contain one or two halogen atoms and/or one or two heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, or phenyl, or wherein the compound of the general formula (I) has the general formula (Ie) ##STR00030## with the following definitions: R.sup.5 hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur. R.sup.6 hydrogen, C.sub.1-12-hydrocarbon residue, which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur. R.sup.7, R.sup.8 independently C.sub.1-8-alkyl, C.sub.3-8-cycloalkyl, which can both contain one or two halogen atoms and/or one or two heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, or phenyl.

7. A method for promoting growth of a plant, comprising adding to growing medium or soil in which the plant is grown, a compound as defined in any claim 1.

8. A mixture containing at least one compound of general formula (I) as defined in claim 1, and at least one additional agrochemical agent, preferably selected from the group consisting of at least one inorganic and/or organic and/or organomineral fertilizer at least one nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBPT) and/or N-n-propylthiophosphoric triamide (NPPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, antisettling agents, coalescing agents, rheology modifiers, defoaming agents, photoprotectors, anti-freeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

9. A fertilizer mixture, containing A. an inorganic and/or organic and/or organomineral fertilizer and B. effective amount, preferably 1 to 10000 weight-ppm, preferably 1 to 100, and even more preferably 1 to 20 weight ppm based on the fertilizer, of a compound of the general formula (I) as defined in claim 1.

10. The fertilizer mixture as claimed in claim 9, wherein the fertilizer mixture is in solid form and the compound of the general formula (I) is incorporated into the fertilizer or is applied to the surface of the, preferably inorganic, fertilizer.

11. The fertilizer mixture as claimed in claim 9, wherein the fertilizer mixture contains at least one additional agrochemical agent, preferably selected from the group consisting of at least one nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBPT) and/or N-n-propylthiophosphoric triamide (NPPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, antisettling agents, coalescing agents, rheology modifiers, defoaming agents, photoprotectors, anti-freeze agents, biostimulants, pesticides/plant production products, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

12. A process for producing the fertilizer mixture as claimed in claim 1 by introducing the compound of the general formula (I) into the fertilizer, and/or applying the compound of the general formula (I) to the surface of the fertilizer.

13. A method of fertilizing soils exploited agriculturally or horticulturally, wherein a fertilizer mixture containing compounds A and B A. an inorganic and/or organic and/or organomineral fertilizer and B. 1 to 10000 weight-ppm, preferably 1 to 100, and even more preferably 1 to 20 weight ppm based on the fertilizer, of a compound of the general formula (I) as defined in claim 1, or compounds A and B separately, but within a period of 0 to 5 hours, preferably 0 to 1 hour, more preferably approximately at the same time, is applied to the soils.

Description

EXAMPLES

[0294] We report on a substance that suppresses Arabidopsis SPX1 and promotes plant growth in phosphate starvation conditions. We validate its positive effect on plant growth in diverse plants, and show that the activity of this substance is retained in a number of variants, which structurally characterize the class of molecules with this activity.

[0295] These results show that the use of SPX genes as a biomarker to screen for SPX suppressing molecules is a potent approach to discover new plant growth promoting or phosphate stress reducing substances.

I. Materials and Methods

1. Plant Material and Growth Conditions

[0296] All Arabidopsis plant experiments were performed using the Arabidopsis thaliana Columbia-0 background. The Arabidopsis pSPX1::GUS line is described in the Plant Journal 2008, 54: 965-975. For rice, the cultivar Nipponbare was used. For maize, the cultivars DKC 2931 and B104 were used for regular pot experiments and sand-based pot experiments, respectively. For soybean, the cultivar Primus was used. Medium-based plant experiments used a modified Murashige and Skoog medium (modMS20 mM NH.sub.4NO.sub.3, 0.1 mM H.sub.3BO.sub.3, 3 mM CaCl.sub.2, 0.1 ?M CoCl.sub.2*6H.sub.2O.sub., 0.1 ?M CuSO.sub.4*5H.sub.2O.sub., 0.1 mM Na.sub.2EDTA*2H.sub.2O.sub., 0.1 mM FeSO.sub.4*7H.sub.2O.sub., 1.5 mM MgSO.sub.4*7H.sub.2O.sub., 0.1 mM MnSO.sub.4*H.sub.2O, 1 ?M Na.sub.2MoO.sub.4*4H.sub.2O.sub., 5 ?M KI, 19 mM KNO.sub.3, 0.03 mM ZnSO.sub.4*7H.sub.2O.sub., 0.1 g/L myo-inositol and 0.5 g/L MES (pH 5,7)), or a regular half strength Murashige and Skoog medium (MS1/2) with 300 ?M phosphate (low phosphate: 300 ?M KH2PO4, 9.9 mM KCl), 600 ?M phosphate (suboptimal phosphate: 600 ?M KH.sub.2PO.sub.4, 9.6 mM KCl) or 1.25 mM phosphate (high phosphate: 1.25 mM KH.sub.2PO.sub.4), except when stated otherwise. The medium was solidified due to the addition of 0.6% Gelrite, unless stated otherwise. In case of vertical plates (root length and root hair measurements), the medium was supplied with 0.5% of sucrose and 1 ?M (low phosphate, 1 ?M KH.sub.2PO.sub.4, 9.9 mM KCl), 10 ?M (low phosphate 10 ?M KH.sub.2PO.sub.4, 9.9 mM KCl) or 1.25 mM KH.sub.2PO.sub.4 (high phosphate). Arabidopsis seeds were stratified for 2 days at 4? C. before germination. Seedlings were germinated in climate-controlled growth chambers at 22? C. under continuous light (100 ?mol m.sup.?2 s.sup.?1). Husked rice seeds were germinated on wetted Whatman paper for 2 days at 37? C. and then transferred to Sterivent boxes or square Petri dishes with solid medium. Maize and soybean seeds were wetted 24 hours at room temperature prior to sowing in pots.

2. Chemical Activity Assessment

[0297] For the chemical activity assessment 3 to 8 seeds per well of Arabidopsis pSPX1::GUS line were sown in 96-well plates containing 150 ?l of low phosphate modMS liquid medium containing 1% of sucrose. Compounds were added in 50 ?M concentration, starting from a 5 mM stock of compounds solved in DMSO. Different chemical substances were separately added to different wells. Per plate, 16 control wells were used, 8 containing only the low phosphate medium and 1% DMSO, and 8 containing high phosphate medium and 1% DMSO. 7 days after germination, plates were scanned using a flatbed scanner (EPSON Expression 11000XL) and analyzed for leaf size. After scanning, the growth medium was replaced by GUS staining solution (see Plant Mol. Biol. Rep. 1994, 12: 37-42 for details) and incubated for 1.5 hours at 37? C. The plates were scanned a second time and analyzed for GUS signal area. Measurements of both leaf area and GUS signal area were conducted by filtering out and measure the green and blue signal, respectively using ImageJ software. If the green leaf area was 50% or less than the control, samples were omitted. Additionally, all results were visually screened for leaf size and GUS signal.

3. Growth Assays

[0298] Arabidopsis primary root growth experiments were done on MS1/2 on vertical standing plates and roots were measured 11 days after germination. Roots were imaged using a flatbed scanner (EPSON Expression 11000XL) and root length was measured using ImageJ software. Arabidopsis shoot biomass was determined in 11 days after germination and growth on vertical standing plates with modMS. Arabidopsis leaf area was measured 21 days after germination on horizontal plates with modMS medium. Plates were scanned from the top using a flatbed scanner (EPSON Expression 11000XL) and the visible leaf area was measured using ImageJ software. Rice plate experiments used vertically standing Petri dishes containing MS1/2 medium (low phosphate0.001 mM) solidified with 0.8% washed agar. Plant height was measured 11 days after germination. Rice box experiments used modMS medium and a low phosphate condition of 0.025 mM phosphate. Plant height was measured 13 days after germination. All growth experiments occurred with 10 ?M of compound and in low phosphate conditions, unless stated otherwise. Controls always received the same DMSO concentration.

4. Pot Experiments

[0299] Regular Arabidopsis, soybean and maize pot experiments used an unfertilized potting soil, which was supplemented at the start of the experiment with 50 ml H.sub.2O containing 0.25 g/l NH.sub.4NO.sub.3 and 0.44 g/l (high phosphate) or no (maize and soybean) or 0.11 g/l KH.sub.2PO (Arabidopsis) for low phosphate conditions. For the sand-based maize pot experiment, sand was supplemented with 50 ml H.sub.2O containing 0.25 g/l NH.sub.4NO.sub.3 and 0.38 g/l (high phosphate) or 0.02 g/l (low phosphate) KH.sub.2PO.sub.4. Potassium was balanced via addition of KCl. Compound concentration was 10 ?M, calculated based on the total water content after watering, and was added at the start of the experiment (Arabidopsis) or once a week (maize). Indexed chlorophyll contents were received via a SPAD 502 Plus Chlorophyll Meter. All parameters in the sand experiment, except the rate of leaf 4 (measured the first 4 days after emergence), were measured 19 days after sowing. Measurements in the pot experiments with potting soil were done after 4 weeks of growth, except for leaf 5, which was measured at 26 days. One long-term maize pot experiment was conducted with the DKC 2931 (2.5 months) and B104 (3 months) cultivars on the PHENOVISION phenotyping platform as described in Frontiers in Plant Science 2021, 12: 640914, except that an unfertilized potting soil was used and that plants were fertilized with a low-phosphate Hoagland solution. The 2.5? overconcentrated low-phosphate Hoagland solution contained 0.034 g/l KH2PO and 200 mL of this solution was applied twice a week. 1.4 mL of a 10 mM compound solution was added each two weeks to each pot, corresponding to 9.6 mg/g nutrients. 13 plants per condition were used. During the experiment, pictures were taken twice a week for each plants from 6 different angles and the top. Pictures were used to extract the plant height and to extract the projected plant area from each side and calculate an estimated plant volume over time.

5. Phosphate Quantification

[0300] Col-0 ecotype Arabidopsis seeds were sown in 96-well plates containing 150 ?l of low phosphate modMS liquid medium containing 1% of sucrose. Compounds was added in 50 ?M concentration (1% DMSO). 7 days after germination, plates were scanned using a flatbed scanner (EPSON Expression 11000XL) and wells containing non-germinated seedlings were excluded from the following analysis. Plants were removed from each well and 50 ?l of medium was recovered from each well and used for molybdenum blue staining as described in Plant Physiology 2008, 146:1673-1686. To measure the phosphate level in soil, Olsen P extraction was used on 1 g of air dried soil, 500 ul of extract was used for molybdenum blue staining as described in The Plant cell 2013, 25:1641-1656. Values were expressed relative to the low phosphate control.

6. Anthocyanin Extraction and Quantification

[0301] Col-0 ecotype Arabidopsis seeds were sown in 96-well plates containing 150 ?l of low phosphate modMS liquid medium containing 1% of sucrose. Compounds was added in 50 ?M concentration (1% DMSO). Eight plants per repeat were collected 7 days after germination and put into a fresh Eppendorf. Extraction and quantification of anthocyanin levels was carried out according to The Plant cell 2013, 25:1641-1656. Anthocyanin levels were quantified by measuring absorption at 530 and 657 nm using a spectrophotometer and normalized to fresh weight input, and expressed relative to the low phosphate control. For pot experiments, the same procedure was followed with one 4-week old plant per sample.

7. Root Hair Length Quantification

[0302] Roots were imaged using a stereomicroscope or a VHX-6000. Number and length of the root hairs parallel to the medium surface was determined at 4 to 6 mm above the root meristem. Root hair length was measured using the ImageJ software.

II. Results

1. Compound 8 Reduces Phosphate Starvation

[0303] To identify new substances that suppress the expression of the SPX1 phosphate starvation biomarker, we used an Arabidopsis SPX1 reporter line pSPX1::GUS. After 7 days of growth in low phosphate conditions, the SPX1 biomarker, visualized by blue GUS staining, is typically clearly induced in the roots. But the molecule 6-amino-1,3-dimethyl-5-[2-(2-methyl-2,3-dihydro-1,4-benzoxazin-4-yl)acetyl]pyrimidine-2,4-dione (named compound 8) practically completely suppressed the SPX1 biomarker, and hence reduces phosphate starvation response. This observation was confirmed in two experimental repeats. 2. The Application of compound 8 reduces phosphate starvation stress Two typical plant responses towards phosphate deficiency are the production of anthocyanin and growth inhibition of the primary root. To confirm the reduction of the phosphate starvation effect by compound 8, its effect on primary root growth and anthocyanin levels were evaluated in Arabidopsis.

[0304] Upon treatment with 10 ?M compound 8, the primary root length of 11-day-old Arabidopsis plants grown in low phosphate is almost double the length of the control without compound (Table 1). Moreover, the anthocyanin level in the leaves is reduced after treatment with compound 8, both when growing the plants in pots or in a 96-well plate assay (Table 1). These observation shows that compound 8 reduces the phosphate starvation response.

TABLE-US-00001 TABLE 1 Parameters related to phosphate starvation stress or growth are affected by compound 8. Control indicates low phosphate conditions without compound (but with compound solvent), unless stated otherwise. Plant and Treatment growth system Parameter Control Compound 8 Arabidopsis Primary root length (cm) 1.8 3.5 vertical plate Shoot biomass (mg) 11 21 Root hair number in high 3 9 phosphate conditions (one plane in 2 mm region) Average root hair length per 0.11 0.32 root (mm) in high phosphate conditions Total root hair length per root 57 183 (cm) in high phosphate conditions Arabidopsis Leaf area (mm.sup.2) 78 106 horizontal plate Leaf area (mm.sup.2) in sub- 104 143 optimal phosphate conditions Arabidopsis pot Rosette size (cm.sup.2) 17 25 Phosphate level in soil 1 0.8 (relative value) Relative anthocyanin level 1 0.6 Arabidopsis Relative anthocyanin level 1 0.6 96-well plate Rice plate Plant height (cm) 14.4 16 Rice box Plant height (cm) 1.1 3.1 Maize sand Plant height (cm) 21 28 Growth rate of leaf 4 (cm/day) 2.04 2.46 Indexed chlorophyll content 28 32 Crown root number 2 7 Maize pot Shoot weight (g) 4 4.6 Plant height (cm) 57 60 Leaf 5 (cm) 40 48 Soybean pot Plant height (cm) 50 55 Biomass (g) 4.1 4.4 Maize pot (long Average number of ears 1 1 term - DKC2931) Shoot weight (g) 27 33 Leaf 6 (cm) 53 58 Maize pot (long Average number of ears 0.3 0.7 term - B104) Shoot weight (g) 28 32 Leaf 6 (cm) 48 50

3. The Application of Compound 8 Leads to Improved Plant Growth

[0305] To further validate the positive effect of compound 8, growth experiments were performed in different growing systems and using different plant species. In different experiments, compound 8 showed a clear positive effect on the shoot biomass, leaf area, formation of ears or plant height in Arabidopsis, rice, soybean and maize, both in plate, box or pot experiments (Table 1). Also in an extensive long-term experiment with two different maize cultivars, compound 8 has a clear promotive effect on growth and on ear setting (Table 1, Table 2). The phosphate levels in the soil after 4 weeks of Arabidopsis growth, shows a reduction if treated with compound 8 (Table 1). This indicates that compound 8 treatment results in higher phosphate uptake.

[0306] Interestingly, we also observed that compound 8 increased both the number of root hairs and the average length of root hairs, which together result in an increased total root hair length (Table 1). Root hairs and the root hair surface are known to be important for phosphate uptake. The induction of the root hairs and the accompanying increase in absorptive area might therefore explain the positive and phosphate starvation reducing effect of compound 8.

TABLE-US-00002 TABLE 2 Estimated volume and plant height based on pictures taken during the long term maize pot experiment for two maize cultivars. Both parameters are clearly promoted by compound 8 (C8) treatment versus control. Estimated volume (cm.sup.3) Plant height (mm) B104 DKC2931 B104 DKC2931 Day C8 Control C8 Control C8 Control C8 Control 15 292 311 393 361 220 230 297 288 19 441 465 763 576 239 246 368 352 22 565 548 1104 843 288 291 372 335 26 689 623 1646 1145 368 351 452 378 27 552 637 1826 1086 364 416 460 352 29 877 788 2187 1581 411 412 471 399 33 1368 1146 3213 2272 428 424 553 424 36 1735 1405 3881 2854 485 472 634 531 40 2117 1673 5092 3872 547 507 694 578 43 2363 1795 5538 4240 587 551 756 655 47 2844 2268 6770 4988 657 609 799 732 50 3518 2874 7419 5718 692 633 844 777 54 3857 3143 8086 6325 564 544 652 562 57 4216 3365 7681 6295 569 572 894 678 61 5052 3936 7458 6047 609 572 1021 893 63 4933 3808 6642 5546 529 514 880 811 68 5837 4585 4967 4292 593 566 895 849 71 5962 4957 4807 4022 629 611 916 856 75 6099 5016 4587 3778 627 569 903 798 78 6540 5551 662 590 82 6751 5788 685 626 85 6623 5957 707 677 89 6434 5717 733 697

4. A Set of Compound 8-Related Molecules Retains Activity and Reduces Phosphate Starvation

[0307] To explore the chemical space of the new phosphate fertilizer enhancer, a variety of structural homologs of compound 8 were characterized by evaluating their effect on the SPX1 phosphate starvation biomarker and/or on root hair development (Table 5). This shows that structures with the general formula (I) retain compound 8 activity and reduce phosphate starvation.

[0308] For a subset of compounds, we performed a more extensive analysis on their root hair inducing effect in a dose-response experiment (Table 3). This confirms that the more simple structures 8.14 and 8.14.6 still retain the activity of compound 8. The higher effect at lower doses moreover suggest a stronger activity. Altogether, this shows that structures with the general formula (I) retain compound 8 activity and reduce phosphate starvation. As all structures induce root hairs and/or suppress the phosphate starvation response, it can be assumed that they, similar to compound 8, have a positive effect on phosphate uptake and hence plant growth in general. To validate this, we performed a maize growth experiment using compound 8 and the, based on the increase of total root hair length, stronger variant 8.14.6. The experiment was conducted in pots with a mixture of unfertilized potting soil and sand, and leaf 5 was measured after 29 days (Table 4). This shows that compound 8.14.6 has even a stronger growth-promoting effect, and confirms that variants of compound 8 that suppress the SPX1 phosphate starvation marker or that induce root hairs or root hair elongation can be expected to have a growth-promoting effect.

TABLE-US-00003 TABLE 3 the effect of different doses of compound 8, 8.14 and 8.14.6 on the total root hair length. Total root hair length per root (in % compared to the control) after Compound treatment with compound at indicated doses code 0.6 ?M 1.25 ?M 2.5 ?M 5 ?M 10 ?M 8 104 333 347 485 586 8.14 231 343 493 803 1074 8.14.6 231 592 1193 1378 1393

TABLE-US-00004 TABLE 4 effect of compounds 8 and 8.14.6 on maize leaf growth in a sand-based pot experiment Compound code Leaf 5 (cm) Control 38.1 8 39.4 8.14.6 41.8

TABLE-US-00005 TABLE 5 Compound 8 and structural variants and their effect on SPX1 suppression (as represented by the GUS stained area) and/or total root hair length. GUS Average GUS Total root (SPX1) (SPX1) stained hair stained area normalized length area nor- to the number per root malized to of seeds of low (in % the num- phosphate con- com- Com- ber of trols in the pared pound seeds same plate to the code Structure (pixels) (pixels) control) 8 [00010] 26 248 657 8.10 [00011] NA 596 8.11 [00012] NA 562 8.12 [00013] NA 341 8.15 [00014] NA 801 8.2 [00015] NA 600 8.7 [00016] NA 641 8.14 [00017] 2 286 884 8.14.1 [00018] 22 1001 172 8.14.2 [00019] 39 1934 455 8.14.3 [00020] 358 8.14.4 [00021] 46 1558 8.14.5 [00022] 93 1385 176 8.14.6 [00023] 2 276 920 NA: data not available