EP2988596A2 - Compounds promoting plant growth - Google Patents

Abstract

The present invention pertains to a method of promoting the growth of a plant and/or improving the stress tolerance of a plant, comprising applying to the plant a benzoic acid derivative. The invention further relates to the use of the benzoic acid derivatives as safeners to protect useful plants from undesired effects of herbicides.

Description

Compounds Promoting Plant growth

BACKGROUND OF THE INVENTION

The present invention relates to the field of crop protection products. It is desirable to identify compounds which are capable of promoting the growth of plants, for example to increase the yield.

When controlling unwanted organisms in crop plant farming which are useful for agriculture or forestry by using pesticides (e.g. herbicides), the useful plants are frequently also damaged to some extent by the pesticides employed. This effect is encountered in particular with the use of a considerable number of herbicides in monocotyledonous and dicotyledonous crops of useful plants. In some instances, the useful plants can be protected against the undesired effects of the herbicides by employing safeners or antidotes, without diminishing the herbicidal activity against the harmful organisms. Only few commercial safeners for dicotyledonous crops have become known. Likewise, for a number of pesticides, hardly any safeners have been described.

It has now been found that, surprisingly, compounds of the formula (I) or (II) shown below or salts thereof can be used effectively as safeners for crop plants or useful plants against damage to these plants caused by agrochemicals, preferably from the group of the selective or non-selective herbicides. The invention thus provides chemical compounds which can be used as safeners. In addition, the compounds exhibit growth-promoting activity on plants.

SUMMARY OF THE INVENTION

The inventors surprisingly found that certain benzoic acid derivatives possess plant growth- promoting activity. It was further found that benzoic acid derivatives can overcome adverse effects of a herbicide on plant growth. The present invention therefore relates to the following subject matter, defined in items (1 ) to (50).

(1 ) A method of regulating plant growth, comprising applying to the plant a compound of Formula (I) or (II)

or a salt thereof,

wherein

n is 0, 1 , 2, 3, 4 or 5,

X is -OR³ or -CO-R¹,

R¹ is -OR³, -NR⁴R⁵, halogen, or an aliphatic group,

each R² is independently selected from the group consisting of amino, nitro, cyano, halogen, alkyl, alkenyl, alkinyl, alkylamino, alkenylamino, alkinylamino, alkoxy, sulfonylamino, alkylsulfonylamino, amino-alkyl, amino-alkenyl, amino-alkinyl, hydroxycarbonyl, aminocarbonyl,

R³ is hydrogen or an optionally substituted aliphatic group,

each of R⁴ and R⁵ is independently selected from the group consisting of hydrogen and optionally substituted alkyl; in particular wherein

n is 0, 1 , 2, 3, 4 or 5,

R¹ is -OR³, -NR⁴R⁵, halogen, or a hydrocarbon chain with 1 to 6 carbon atoms, each R² is independently selected from the group consisting of amino, nitro, cyano, halogen, (C_rC₆)-alkyl, (C₂-C₆)-alkenyl, (C₂-C₆)-alkinyl, (C C_e)-alkylamino, (C₂-C₆)- alkenylamino, (C₂-C₆)-alkinylamino, (C C₆)-alkoxy, sulfonylamino, (C C₆)- alkylsulfonylamino, amino-(C C₆)-alkyl, amino-(C₂-C₆)-alkenyl, amino-(C₂-C₆)-alkinyl, hydroxycarbonyl, aminocarbonyl,

R³ is hydrogen or optionally substituted (C C₆)-alkyl,

each of R⁴ and R⁵ is independently selected from the group consisting of hydrogen and optionally substituted (C C₆)-alkyl.

(2) A method of promoting plant growth, comprising applying to the plant a compound of Formula (I) or (II) or a salt thereof as defined in item (1 ).

(3) The method of item (1 ) or (2), further comprising applying a herbicide to the plant. (4) The method of item (3), wherein the herbicide is glyphosate, glufosinate, chlorophenoxyacetic acid, pethoxamide, amidosulfuron.sulfuronmethyl, imidazoline or others.

(5) A method of improving the stress tolerance of a plant, comprising applying to the plant a compound of Formula (I) or (II) or a salt thereof as defined in item (1 ). (6) The method item (5), wherein the stress tolerance is tolerance to one or more herbicides.

(7) The method of item (5) or (6), wherein the plant has been treated, is treated or is to be treated with a herbicide.

(8) The method of item (7), wherein the herbicide is glyphosate, glufosinate, chlorophenoxyacetic acid, pethoxamide, amidosulfuron.sulfuronmethyl, imidazoline or others.

(9) The method of item (5), wherein the stress is abiotic stress.

(10) The method of item (9), wherein the stress tolerance is tolerance to high or low temperature, or to droughtness.

(1 1 ) The method of any one of the preceding items, wherein n is 0, 1 , 2, 3 or 4. (12) The method of any one of the preceding items, wherein n is 0, 1 , 2, or 3.

(13) The method of any one of the preceding items, wherein n is 0, 1 or 2.

(14) The method of any one of the preceding items, wherein n is 0 or 1.

(15) The method of any one of the preceding items, wherein n is 1 .

(16) The method of any one of items (1 ) to (14), wherein n is 0. (17) The method of item (16), wherein the compound is benzoic acid (BA).

(18) . The method of any one of the preceding items, wherein R¹ is -OH or -NH₂.

(19) The method of any one of the preceding items, wherein R¹ is -OH.

(20) The method of any one of the preceding items, wherein each R² is independently selected from the group consisting of amino, cyano, and methyl. (21 ) The method of any one of items (1 ) to (15) and (18) to (20), wherein each R² is amino.

(22) The method of item (21 ), wherein n is 1 .

(23) The method of any one of items (1 ) to (15) and (18) to (20), wherein one group R² is attached to the carbon atom at position 4 of the aromatic ring.

(24) The method of item (23), wherein n is 1.

(25) The method of any one of items (1 ) to (15) and (18) to (24), wherein the compound is 4-aminobenzoic acid (pABA).

(26) The method of any one of items (1 ) to (15) and (18) to (20), wherein one group R² is attached to the carbon atom at position 3 of the aromatic ring and/or n is 1.

(27) The method of any one of items (1 ) to (26), wherein the pK_a value of the compound is at least 4.0, preferably at least 4.5, most preferably at least 5.0.

(28) The method of item (27), wherein the compound is 3-aminobenzoic acid (mABA).

(29) The method of any one of the preceding items, wherein the compound of Formula (I) or (II) or a salt thereof is applied at a concentration of from 1 μΜ to 100 mM, preferably of from 100 μΜ to 10 mM.

(30) The method of any one of the preceding items, wherein the compound is applied to the roots of the plant, preferably by watering the cultured area.

(31 ) The method of any one of items (1 ) to (29), wherein the compound is applied to the foliage of the plant.

(32) The method of item (31 ), wherein the compound is sprayed onto the foliage of the plant.

(33) The method of any one of the preceding items, further comprising applying a plant growth regulator to the plant which is different from the compound of Formula (I) or (II) or a salt thereof.

(34) The method of any one of the preceding items, wherein the compound is applied to the plant once per day. (35) The method of any one of the preceding items, wherein the total daily dose of the compound per plant is in the range from 1 μιτιοΙ to 100 mmol, preferably from 10 μιηοΙ to 10 mmol, most preferably from 50 pmol to 1 mmol.

(36) The method of any one of the preceding items, wherein the plant is selected from the group consisting of corn, rice, wheat, barley, rye, oat, sorghum, cotton, soybean, peanut, buckwheat, beet, oilseed rape, sunflower, sugar cane, tobacco, and hop.

(37) The use of a compound of Formula (I) or (II) or a salt thereof as defined in any one of items (1 ) to (28) as a safener.

(38) The use of a compound of Formula (I) or (II) or a salt thereof as defined in any one of items (1 ) to (28) as a plant growth regulator.

(39) The use of a compound of Formula (I) or (II) or a salt thereof as defined in any one of items (1 ) to (28) as a plant growth promoter.

(40) An agrochemical composition comprising a compound of Formula (I) or (II) or a salt thereof as defined in any one of items (1 ) to (28). (41 ) A plant safener composition comprising the compound of Formula (I) or (II) or a salt thereof as defined in any one of items (1 ) to (28).

(42) The composition of item (40) or (41 ), comprising the compound of Formula (I) or (II) or a salt thereof at a concentration of 10 μΜ to 10 mM.

(43) The composition of item (42), comprising the compound of Formula (I) or (II) or a salt thereof at a concentration of 100 μΜ to 1 mM.

(44) The composition of item (43), comprising the compound of Formula (I) or (II) or a salt thereof at a concentration of 500 μΜ to 1 mM.

(45) The composition of any one of items (40) to (44), further comprising a herbicide.

(46) The composition of item (44), wherein the herbicide is selected from the group consisting of glyphosate, glufosinate, chlorophenoxyacetic acid, pethoxamide, amidosulfuron, sulfuronmethyl, imidazoline or others.

(47) A method of detecting a receptor molecule binding to a compound of Formula (I) or (II) or a salt thereof as defined in any one of items (1 ) to (28), comprising (i) attaching the compound of Formula (I) or (II) or a salt thereof to a solid support to obtain a modified solid support, (ii) contacting the modified solid support with homogenized plant material, (iii) identifying and optionally characterizing plant molecules bound to the modified solid support.

(48) The method of item (47), wherein the compound of Formula (I) or (II) is p- aminobenzoic acid (pABA).

(49) A method of determining the biologic activity of a compound of Formula (I) or (II) as defined in any one of items (1 ) to (28), comprising providing the compound of Formula (I) or (II) or a salt thereof and determining its plant growth promoting activity and/or its stress tolerance conferring activity.

(50) A method of screening for plant growth-promoting compounds and/or plant safeners, comprising providing a derivative of a compound of Formula (I) or (II) as defined in any one of items (1 ) to (28), and determining the plant growth promoting activity and/or the stress tolerance conferring activity of the derivative.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. pABA promotes lateral root, adventitious root and root hairs development. A-B, 10 day-old wild type Arabidopsis thaliana (L.) Heynh. ectoype Columbia (Col-0), grown in presence or absence of 200 μΜ pABA. A, Root hairs, adventitious and lateral root development. B, quantitation of lateral root development. C, Lycopersicum esculentum (tomato) grown in presence or absence of pABA. White arrows indicate adventitious roots, black arrows indicate lateral roots. Asterisk (*) indicates significant difference t>95%.

Figure 2. pABA promotes plant biomass formation when applied to Arabidopsis thaliana.

Figure 3.pABA promotes plant biomass formation when applied to Zea mays. Asterisk (*) indicates significant difference t>95%.

Figure 4. Visualization of tissue specific production and metabolization of para-aminobenzoic acid (pABA). (A) Folate pathway with pABA synthesis by GAT-ADCS (*), esterification by UGT75B (**) and fusion to pterin by HPPK-DHPS (***). (B) Schematic representation of pABA. (C) Tissue expression of GAT-ADCS and HPPK-DHPS in Arabidopsis thaliana seedlings. GAT-ADCS and HPPK-DHPS are coexpressed in the same tissues. (D) GAT- ADCS and HPPK-DHPS expression in the root tip. Left, for short time staining (30 min), GAT-ADCS was expressed in columella and lateral root cap cells (#). Middle, when stained for longer time (3h), GAT-ADCS expression domain is extended to epidermal (white *) and cortex (black *) cells. Right, HPPK-DHPS promoter activity. HPPK-DHPS promoter activity globally matches GAT-ADCS promoter expression pattern. Figure 5. Expression of GAT-ADCS in pollen and female gametophyte during early embryogenesis. (A) pGAT-ADCS::GUS signal in pollen grains. (B) GAT-ADCS expression in female gametophyte. (C) GAT-ADCS expression in seed with embryo at beginning of globular stage. (D) Enlargement of boxed area in C. Note endosperm-derived GUS signal (*) surrounding the embryo. (E) GAT-ADCS expression in seed with embryo at late globular stage. Note drastic reduction of GAT-ADCS expression in endosperm, while GUS signal has appeared in embryo. (F) Enlargement of boxed area in E. (G) GAT-ADCS expression in seed with embryo at heart stage. (H) Enlargement of boxed area in G.

Figure 6. Characterization of GAT-ADCS. (A) GAT-ADCS (At2g28880) gene model (6.5 kb) including promoter, UTR, introns, exons and T-DNA insertions. (B) T-DNA insertion in adcs2 mutant shown by PCR. (C) Abnormal seeds (*) in opened siliques in adcs2. (D) Seeds ratio indicating wild-type (WT)-like and defective seeds in adcs2. (E) Globular stage arrest of adcs2 embryos (arrowed). (F) Complementation of adcs2 (line 10, 1 1 and 17) with WT ADCS sequence shown by PCR, T-DNA is present in adcs2 but absent in WT, while pGAT- ADCS::ADCS construct can be detected in mutants but not in WT. (G) Confirmation of complementation of lines 10, 1 1 and 17 with pGAT-ADCS::ADCS. WT GAT-ADCS sequence complements adcs2 globular stage arrest, shown by a drop of defective seeds from 25% to 6.25% for the lines 10 and 17. Line 1 1 probably contains double pGAT-ADCS::ADCS /Insertions, indicated by a drop of defective embryos from 25% to 1.56%. Scale bar = 500μιη (C) and 100pm (E). Figure 7. Characterization of adcs-1. WT and adcs-1 seedlings growing on AM medium. Scale bar=1cm.

Figure 8. Homozygous adcs2 rescue experiment with pABA. (A) adcs2 opened silique incubated on ppABA supplemented medium. (B) Embryo development and germination 3 weeks later. (C) One week after germination, difference in growth become apparent (left WT, right adcs knockout). (D) Magnified view of boxed area in (C) 3 weeks after germination. Note that shoot parts (arrow) not in contact with pABA supplemented medium do not develop. Scale bar = 100 pm in A, B, D and 1 cm in C. Figure 9. Conditional down regulation of ADCS. (A) 20μΜ estradiol (EST20) induction results in down regulation of GAT-ADCS expression. (B) EST application induces root growth arrest of 9 day-old /ADCS-RNAi seedlings. (C) Growth arrest can be rescued by growing plants for 6 days in either 50μΜ pABA or 50μΜ 5-formyl tetrahydrofolate (5-FTHF). The letters indicate independent groups according to one-way analysis of variance test (C). Scale bar=1 cm. Mock (AM + ethanol).

Figure 10. Regulation of plant growth by exogenous application of pABA. (A) Root growth of 6 day-old Arabidopsis thaliana in presence of increasing concentrations of pABA. Root growth is repressed by pABA. (B) Arabidopsis seedlings growing on AM, 100μΜ pABA and 100μΜ 5-FTHF supplemented media. (C) Percentage of emerged adventitious (AR) and lateral (LR) roots on AM, pABA and 5-FTHF supplemented media. pABA induces development of LR and AR. (D) Stimulation of root hair elongation by pABA. (E) Quantification of root hair size. pABA treatment increases root hair length. (F) Root growth of 6 day-old Arabidopsis grown in presence of 100μΜ pABA, 100μΜ 5-FTHF and 100μΜ of the folate synthesis inhibitor Sulfanilamide (Sulf). Root growth inhibition by Sulf is reverted by both 5-FTHF and pABA. (G) Root response to gravity after rotating plates bearing roots to 135° for 3h in AM, pABA, and 5-FTHF supplemented media. pABA increases gravitropic response of Arabidopsis primary root. Scale bars = 0.5 cm (b) and 500 pm (f). The asterisk indicates a significant difference from control by Student's test (p< 0.01 )(E).The letters indicate independent groups according to one-way analysis of variance test (F).

Figure 11. Quantification of free pABA and pABA-Gluc in Arabidopsis seedlings. (A) pABA in WT. (B) pABA in ugt75B mutant. (C) pABA in non-induced Lex::UGT75B. (D) pABA in Lex::UGT75B seedlings induced with 1 μΜ estradiol. With estradiol concentration higher than 1 μΜ the growth is limited and it is difficult to get enough plant material for pABA determinations. Each value is the average of three independent experiments ±SD. The asterisk indicates a significant difference from control by Student's test (p< 0.01 ).

Figure 12. GAT-ADCS, HPPK-DHPS and UGT75B expression domains in Arabidopsis root tip. (A) GAT-ADCS, (B) HPPK-DHPS expression pattern. (C) UGT75B expression pattern. UGT75B::3xYFP is expressed in whole root tip except in the stele, columella cells and the QC. (D) Close-up of root presented in C. UGT75B::3xYFP signal is mainly present in lateral root cap (LRC), epidermis (Ep) and cortex (co) cells. Scale bars = 50pm (in A, B, C), 20μηι (in D). Figure 13. Impact of pABA-conjugation on root development. (A-B) Quantification of adventitious root (AR) and root length of ugt75b mutant and WT in presence of pABA. (A) AR, (B) root length. ugt75b roots are more sensitive to pABA. (C) Induction of Lex::UGTB75B with EST followed by RT-PCR. (D) Severe root growth defects after strong induction of Lex::UGTB75B with 20μΜ EST. (E) Root growth phenotype upon weak induction of Lex::UGTB75B with 0.1 μ EST in presence of 200 μΜ pABA and 200μΜ 5-FTHF. pABA rescues effects of over expressed Lex::UGTB75B but not 5-FTHF. (F) Quantification of root slanting in presence of pABA and 5-FTHF respectively, after EST-weak induction of Lex::UGT75B. (G) Quantification of root growth in presence of pABA and 5-FTHF after EST- weak induction of Lex::UGT75B. Mock (AM +ethanol).The asterisk indicates a significant difference from control by Student's test (p< 0.05)(B). The letters indicate independent groups according to one-way analysis of variance test (F-G).

Figure 14. Impact of pABA conjugation on root development. Arabidopsis seedlings growing for 9 days in presence of 20μΜ EST. (A) Strong EST induction ofLex::UGTB75B results in severe root growth defects which can be only partially rescued by 400μΜ pABA and very poorly by 5-FTHF applied at the same concentration. (B) Quantification of root length. Mock (AM + ethanol).The letters indicate independent groups according to one-way analysis of variance test (B). Scale bar = 1 cm.

Figure 15. Root development and auxin distribution. (A) Root development of WT and Lex::UGT75B seedlings grown in presence of EST, or EST supplemented with 250μΜ pABA or 1 mM5-FTHF. (B) PIN1 (red) and PIN2 (green) subcellular localization in roots of WT and Lex::UGT75B grown in presence of EST, or EST supplemented with 250μΜ pABA, 1 mM5- FTHF. Epidermis (Ep), Cortex (Co), Endodermis (En).Scale bar = 20 μιη.

Figure 16. Auxin response in root tips of pDR5.:GL/SandLex::L/GT75S transgenic plants. (A- B) pDR5::GUS plants displaying typical auxin maximum in QC and columella cells. (A) Mock (AM+ethanol) treated pDR5::GUS plants. (B) pDR5;;GL/Splants transferred for 48 hours to growth medium supplemented with 5μΜ EST. (C-D) pDR5::GUS in Lex::UGT75B transgenic plants. (C) Non induced and (D) after induction with 5μΜ EST. Note ectopic DR5 signal in LRC initials (arrowed).

Figure 17. Safener activity of pABA on Arabidospsis thaliana seedlings grown in presence of inhibitory concentrations of auxins showing antagonism of pABA with auxins. Lower panel: quantification of root length and structures of molecules used (Example 5). Figure 18. Safener activity of benzoic acid derivatives on Arabidospsis thaliana seedlings grown in presence of inhibitory concentrations of auxins (Example 5).

Figure 19. Activity of benzoic acid derivatives on Arabidopsis root development (Example 5).

Figure 20. Confirmation of safener activity of pABA (Example 6). Arabidopsis thaliana Auxin reporter DR5::GFP plants incubated in presence of 1 μΜ IAA, 100μΜ PABA, 10μΜ NPA applied either separately or in combinations. (A) Seedlings grown in presence of chemicals. (B) Quantification of root length of seedlings presented in (A). (C) Visualization of DR5::GFP signal from root tips of plant presented in (A).

DETAILED DESCRIPTION

Definitions

The term "aliphatic group", as used herein, includes saturated or unsaturated, branched or unbranched aliphatic monovalent or bivalent groups. In the present application, aliphatic group is intended to include, but is not limited to, alkyl, cycloalkyl, alkenyl, and alkinyl groups. According to the present invention, the aliphatic group has 1 to 100, preferably 1 to 42 carbon atoms, preferably 1 to 22 carbon atoms, more preferred 1 to 15 carbon atoms, further preferred 1 to 10 carbon atoms, even more preferred 1 to 6 carbon atoms, for instance 1 , 2, 3 or 4 carbon atoms. Most preferably, the aliphatic group is Ci_₆-alkyl, e.g. methyl, ethyl, propyl and butyl. Aliphatic groups as used herein, include heteroaliphatic groups in which one or more carbon atoms have been substituted with a heteroatom, for instance, with an oxygen, sulfur, nitrogen, phosphorus or silicon atom, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroaliphatic group. Examples include -CH₂-CH₂-0-CH₃, -CH₂-CH₂-NH-CH₃, -CH2-CH₂-N(CH₃)-CH3, -CH₂-S-CH₂- CH₃, ~S(0)-CH₃, -CH₂-CH₂-S(0)₂-CH₃, -CH=CH-0-CH₃, -CH₂-CH=N-OCH₃, and -CH=CH- N(CH₃)-CH₃. A heteroaliphatic group may be linear or branched, and saturated or unsaturated. The term "alkyl" used is the present application relates a saturated branched or unbranched aliphatic monovalent substituent. Preferably, the alkyl group has 1 to 100 carbon atoms, more preferred 1 to 22 carbon atoms, further preferred 1 to 10 carbon atoms, yet more preferred 1 to 6 carbon atoms, even more preferred 1 to 3 carbon atoms. Accordingly, examples of the alky] group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, terf-butyl, n-pentyl and n-hexyl and preferable examples include methyl, ethyl, n-propyl and isopropyl, whereby ethyl and isopropyl are particularly preferred. As used herein, the term "cycloalkyl" refers to a monocyclic, bicyclic, or tricyclic group, which may be saturated or partially saturated, i.e. possesses one or more double bonds. Monocyclic groups are exemplified by a saturated cyclic hydrocarbon group containing from 3 to 8 carbon atoms. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl. Bicyclic fused cycloalkyl groups are exemplified by a cycloalkyl ring fused to another cycloalkyl ring. Examples of bicyclic cycloalkyl groups include, but are not limited to decalin, 1 ,2,3,7,8,8a-hexahydro-naphthalene, and the like. Tricyclic cycloalkyl groups are exemplified by a cycloalkyl bicyclic fused ring fused to an additional cycloalkyl group. The term "alkenyl" as used is the present application is an unsaturated branched or unbranched aliphatic monovalent group having at least one double bond between two adjacent carbon atoms. Preferably, the alkenyl group has 2 to 6 carbon atoms, more preferred 2 to 4 carbon atoms. Accordingly, examples of the alkenyl group include but are not limited to ethenyl, 1 -propenyl, 2-propenyl, methylvinyl, 1-butenyl, 2-butenyl, 3-butenyl, 2- methyl-1 -propenyl, 2-methyl-2-propenyl, 2-pentenyl, 2-hexenyl. Preferable examples of the alkenyl group include ethenyl, 1 -propenyl and 2-propenyl, whereby ethenyl is particularly preferred. The term "alkenyl" includes alkadienyl groups having two double bonds. Preferably, the alkadienyl group has 4 to 10 carbon atoms. Accordingly, examples of the alkadienyl group include but are not limited to 2,4-pentadienyl, 2,4-hexadienyl, 4-methyl-2,4- pentadienyl, 2,4-heptadienyl, 2,6-heptadienyl, 3-methyl-2,4-hexadienyl, 2,6-octadienyl, 3- methyl-2,6-heptadienyl, 2-methyl-2,4-heptadienyl, 2,8-nonadienyl, 3-methyl-2,6-octadienyl, 2,6-decadienyl, 2,9-decadienyl and 3,7-dimethyl-2,6-octadienyl groups, whereby 2,4- pentadienyl is particularly preferred. The term "alkinyl" as used is the present application is an unsaturated branched or unbranched aliphatic monovalent group having at least one triple bond between two adjacent carbon atoms. Preferably, the alkinyl group has 2 to 6 carbon atoms, more preferred 2 to 4 carbon atoms. Examples of the alkinyl group include but are not limited to ethinyl, 1-propinyl, 1-butinyl, 2-butinyl, 1-pentinyl, 2-pentinyl, 3-pentinyl and 2-hexinyl. Preferable examples of the alkenylene group include ethinyl, 1 -propinyl and 2-propinyl, whereby ethinyl is particularly preferred. The terms "halo" and "halogen" refer to a halogen atom selected from the group consisting of F, CI, Br and I. Preferably the halogen atom is CI or Br, whereby CI is particularly preferred. The term "halogenated alkyi group" refers to an alkyi groups as defined above which is substituted with at least one halogen atom. In a preferred embodiment, the halogenated alkyi group is perhalogenated. In a more preferred embodiment, the halogenated alkyi group is a univalent perfluorated group of formula C_nF_2n+i . Preferably, the halogenated alkyi group has 1 to 6 carbon atoms, even more preferred 1 to 3 carbon atoms. Accordingly, examples of the alkyi group include trifluoromethyl, 2,2,2-trifluoroethyl, n-perfluoropropyl, n-perfluorobutyl and n-perfluoropentyl. Preferable examples of halogenated alkyi groups include trifluoromethyl and 2,2,2-trifluoroethyl, whereby trifluoromethyl is particularly preferred.

The pK_a value is equal to -log₁₀ K_a, wherein K_a is the dissociation constant which defines the ratio of the concentrations of the dissociated ions and the undissociated acid:

_ [A-] [H+]

[HA]

Methods of determining the pK_a for a compound will be readily understood by the skilled artisan. Some compounds include more than one acidic hydrogen and therefore have more than a single pK_a value. According to the invention, the relevant and referred to pK_a is the highest pK_a of a compound. If the compound is a compound of Formula (I) wherein R is OH, then the pK_a value preferably refers to the pK_a value of the carboxylic group -CO-R¹. If the compound is a compound of Formula (II) wherein X is COOH, then the pK_a value preferably refers to the pK_a value of the carboxylic group X.

The term 'safener', as used herein, refers to compounds which compensate for, or reduce, the phytotoxic properties of a pesticide (e.g. a herbicide) toward useful plants without essentially reducing the herbicidal action against harmful plants.

The present invention relates to a method of promoting the growth of a plant and/or improving the stress tolerance of a plant. The method preferably comprises applying to the plant a compound of Formula (II)

wherein

n is O, 1 , 2, 3, 4 or 5,

X is -OR³ or -CO-R¹,

R¹ is -OR³, -NR⁴R⁵, halogen, or an aliphatic group,

each R² is independently selected from the group consisting of amino, nitro, cyano, halogen, alkyl, alkenyl, alkinyl, alkylamino, alkenylamino, alkinylamino, alkoxy, sulfonylamino, alkylsulfonylamino, amino-alkyl, amino-alkenyl, amino-alkinyl, hydroxycarbonyl, aminocarbonyl, halogenated alkyl, aryl, heteroaryl,

R³ is hydrogen or an optionally substituted aliphatic group,

each of R⁴ and R⁵ is independently selected from the group consisting of hydrogen and optionally substituted alkyl.

In a preferred embodiment, the compound is a compound of Formula (I)

or a salt thereof,

wherein n, R¹, R², R³, R⁴ and R⁵ are defined as above for Formula (II).

The parameter n may be 0, 1 , 2, 3, 4 or 5. Preferably, n is selected from 0, 1 , 2, 3, and 4; more preferably n is selected from 0, 1 , 2, and 3; still more preferably, n is selected from 0, 1 , and 2; most preferably n is 1 . Group X of formula (II) can be -OR³ or -CO-R¹. Preferably, group X is -CO-R¹, as indicated in Formula (I).

R¹ is typically selected from the group consisting of -OR³, -NR R⁵, halogen, and an aliphatic group. Preferably, R¹ is -OR³. R³ can be hydrogen or an optionally substituted aliphatic group. Preferably, R³ is hydrogen or an optionally substituted alkyl group; more preferably, R³ is hydrogen or an optionally substituted C C₆ alkyl group. Still more preferably, R³ is hydrogen or C C₆ alkyl. Most preferably, R³ is hydrogen. Accordingly, group X most preferably is -COOH.

Group R² is, for each occurrence of R², independently selected from the group consisting of amino, nitro, cyano, halogen, alkyl, alkenyl, alkinyl, alkylamino, alkenylamino, alkinylamino, alkoxy, sulfonylamino, alkylsulfonylamino, amino-alkyl, amino-alkenyl, amino-alkinyl, hydroxycarbonyl, aminocarbonyl, halogenated alkyl, aryl, heteroaryl.

Preferably, group R² is selected from the group consisting of amino, nitro, cyano, halogen, (C₁-C₆)-alkyl, (C₂-C₆)-alkenyl, (C₂-C₆)-alkinyl, (C -alkylamino, (C₂-C₆)-alkenylamino, (C₂- C₆)-alkinylamino, (C C₆)-alkoxy, sulfonylamino, (C C₆)-alkylsulfonylamino, amino-(C C₆)- alkyl, amino-(C₂-C₆)-alkenyl, amino-(C₂-C₆)-alkinyl, hydroxycarbonyl, aminocarbonyl.

More preferably, group R² is selected from alkyl, amino and aminoalkyl; still more preferably, group R² is selected from amino and C C₆ alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n- butyl, fe/f-butyl etc. Most preferably, group R² is amino. In one embodiment, the compound of the invention comprises at least one substituent R² in para position (position 4 of the phenyl ring), e.g. the compound 4-aminobenzoic acid. In another embodiment, the compound of the invention comprises at least one substituent R² in meta position (position 3 of the phenyl ring), e.g. the compound 3-amino benzoic acid. In yet another embodiment, the compound of the invention comprises at least one substituent R² in ortho position (position 2 of the phenyl ring), e.g. the compound 2-amino benzoic acid.

In yet another embodiment, n is 1 and group R² is bound to the phenyl ring in para position. In another embodiment, n is 1 and group R² is bound to the phenyl ring in meta- or ort o- position.

In a specific embodiment n is 2 and each of the two groups R² is bound to the phenyl ring in /nefa-position (positions 3 and 5 of the phenyl ring).

In one embodiment of the invention the compound of the invention is a compound of Formula (III) OH

(^R2)" (III) or a salt thereof, wherein n and R² have the same meaning as defined above. r embodiment, the compound of the invention is a compound of Formula (IV) (IV) or a salt thereof, wherein R² has the same meaning as defined above.

r embodiment, the compound of the invention is a compound of Formula (V) (V) or a salt thereof, wherein R² has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (VI) (VI) or a salt thereof, wherein R² has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (VII) (VII) or a salt thereof, wherein R² has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (VIII) (VIII) or a salt thereof, wherein R² has the same meaning as defined above.

embodiment, the compound of the invention is a compound of Formula (IX) (IX) or a salt thereof, wherein R¹ has the same meaning as defined above. In another embodiment, the compound of the invention is a compound of Formula (X) x

(NH_)_n (x) _{or a sa}|t thereof, wherein n and X have the same meaning as defined above. In another embodiment, the compound of the invention is a compound of Formula (XI) rr^CH3 (XI) or a salt thereof, wherein R¹ has the same meaning as defined above. ther embodiment, the compound of the invention is a compound of Formula (XII) (XII) or a salt thereof, wherein R¹ has the same meaning as defined above.

her embodiment, the compound of the invention is a compound of Formula (XIII) (XIII) or a salt thereof, wherein R¹ has the same meaning as defined above,

embodiment, the compound of the invention is a compound of Formula (XIV) (XIV) or a salt thereof, wherein R¹ has the same meaning as defined above.

embodiment, the compound of the invention is a compound of Formula (XV) (XV) or a salt thereof, wherein R¹ has the same meaning as defined above. In another embodiment, the compound of the invention is a compound of Formula (XVI) (XVI) or a salt thereof, wherein R¹ has the same meaning as defined above.

her embodiment, the compound of the invention is a compound of Formula (XVII) (XVII) or a salt thereof, wherein R¹ has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XVIII) (XVIII) or a salt thereof, wherein n and R have the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XIX) (xix) or a salt thereof, wherein n and R¹ have the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XX) (XX) or a salt thereof, wherein X has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XXI) (XXI) or a salt thereof, wherein X has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XXII) (XXII) or a salt thereof, wherein X has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XXIII) (XXIII) or a salt thereof, wherein X has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XXIV) (XXIV) or a salt thereof, wherein X has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XXV)

(XXV) or a salt thereof, wherein X has the same meaning as defined above.

In another embodiment, the compound of the invention is a compound of Formula (XXVI) (XXVI) or a salt thereof, wherein X and n have the same meaning as defined above.

It is preferred that the compounds of the invention have at least one acidic hydrogen atom which can dissociate as H⁺ in dependence of the pH.

Preferably, the compound in accordance with this invention has a pK_a value of at least 4.0, preferably of at least 4.25, more preferably of at least 4.5, still more preferably of at least 4.75, most preferably of at least 5.0. Typically, the pK_a value ranges from 4.0 to 10.0, preferably from 4.5 to 8.0, most preferably from 5.0 to 7.0. The inventors surprisingly found that there is a correlation between the pK_a value of the compound and its activity as plant growth promoter and safener. Compounds having a pK_a value of at least 5 turned out to be most active. Examplary compounds of the invention include, but are not limited to, the following compounds and salts thereof:

2-Methylbenzoic acid; 2-Ethylbenzic acid; 3-Methylbenzoic acid; 3-Ethylbenzic acid; 4- Methylbenzoic acid; 4-Ethylbenzic acid; 3,5-Dimethylbenzoic acid; 3,5-Diethylbenzoic acid; 2,6-Dimethylbenzoic acid; 2,6-Diethylbenzoic acid;

3,5-Di-ierf-butylbenzoic acid, 2-terf-butylbenzoic acid, 3-ferf-butylbenzoic acid, 4-tert- butylbenzoic acid;

BiphenyI-4-carboxylic acid;

4-(Methylthio)benzoic acid; 3-(Methylthio)benzoic acid; 2-(Methylthio)benzoic acid;

2-Methoxybenzoic acid; 3-Methoxybenzoic acid; 4-Methoxybenzoic acid; 3,5- Dimethoxybenzoic acid; 3,4,5-Trimethoxybenzoic acid;

2-lsopropylbenzoic acid; 3-lsopropylbenzoic acid; 4-lsopropylbenzoic acid; 3,5-Di- isopropylbenzoic acid;

3-Hydroxybenzoic acid, 4-Hydroxybenzoic acid, 3,4-Dihydroxybenzoic acid, 3,5- Dihydroxybenzoic acid, 3,4,5-Trihydroxybenzoic acid;

4-Aminobenzoic acid, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 3,5-Diaminobenzoic acid, 3,4-Diaminobenzoic acid;

4-(Methylamino)benzoic acid, 2-(Methylamino)benzoic acid, 3- (Methylamino)benzoic acid, 3,5-Di-(methylamino)benzoic acid, 3,4-Di-(methylamino)benzoic acid, procaine;

4-(Dimethylamino)benzoic acid, 2-(Dimethylamino)benzoic acid, 3-(Dimethylamino)benzoic acid, 3,5-Di-(methylamino)benzoic acid, 3,4-Di-(methylamino)benzoic acid.

Further active compounds of the invention (CAS numbers in parentheses, where applicable) include 4-Nitrophenol, 3-Nitrophenol, 2-Nitrophenol, Ascorbic acid, Dimedon, Meldrums acid, Dinitropyrazol, 3-Nitro-1 ,2,4-triazol, Benzotriazol, Acetylsalicylic acid, Chlorothiazide, Chloroazepine acid, Cyclobutane carboxylic acid, Cyclohexandion-1 ,3, Diclofenac, Piperonylic acid, 2,3-dihydro-1 ,4-benzodioxine-6-carboxylic acid (4442-54-0), 1 ,2,3,4- tetrahydro-6-quinolinecarboxylic acid (5382-49-0), 4-morpholinobenzoic acid (7470-38-4), 4- (1 H-imidazol-1 -yl)benzoic acid (17616-04-5), Isonipecotic acid, 4-pyridyl carboxylic acid, 2- methyl-6-quinolinecarboxylic acid (635-80-3), 2-Methyl-1 H-benzimidazole-5-carboxylic acid (709-19-3), lndole-5-carboxylic acid (1670-81 -1 ), and 1-benzothiophene-5-carboxylic acid (2060-64-2). Another aspect of the invention is the use of a compound as defined herein as a plant growth promotor. The invention also provides a method for protecting crop plants or useful plants against phytotoxic actions of agrochemicals, such as herbicides, which method comprises using compounds of the formula (I) or (II) or salts thereof as safeners, preferably by applying an effective amount of the compounds of the formula (I) or (II) or salts thereof to the plants, to parts of plants or seeds thereof.

For the joint use with agrochemicals/herbicides, the compounds of the formula (I) or (II) according to the invention or salts thereof can be applied simultaneously with the active compounds or in any order, and they are then capable of reducing or completely eliminating harmful side effects of these active compounds in crop plants, without negatively affecting or substantially reducing the activity of these active compounds against unwanted harmful organisms. Here, even damage caused by using a plurality of herbicides, insecticides or fungicides, or herbicides in combination with insecticides or fungicides, can be reduced substantially or eliminated completely. In this manner, it is possible to extend the field of use of conventional pesticides considerably.

If the agrochemical compositions according to the invention comprise herbicides, these compositions are, after appropriate dilution, applied either directly to the area under cultivation, to the already germinated harmful and/or useful plants or to the already emerged harmful and/or useful plants. If the compositions according to the invention do not comprise any herbicide, these compositions can be employed by the tank mix method (i.e. the user mixes and dilutes the separately available products (the herbicide and the safener) immediately prior to application to the area to be treated, or prior to the application of a herbicide, or after the application of a herbicide, or for the pretreatment of seed, for example, for dressing the seed of the useful plants.

The compounds according to the invention can be applied together with the herbicides by the pre-emergence method or the post-emergence method, for example in the case of simultaneous application as a tank mix or a co-formulation or in the case of a separate application, in parallel or in succession (split application). It is also possible to repeat the application a number of times. In some cases, it may be expedient to combine a pre- emergence application with a post-emergence application. In most cases, one option is a post-emergence application to the useful plant or crop plant together with a simultaneous or later application of the herbicide. Also possible is the use of the compounds according to the invention or salts thereof for seed dressing, for (dip) treatment of seedlings or for the treatment of other propagation material (for example potato tubers).

Herbicides whose undesired side effects on useful plants can be reduced using compounds of the formula (I) or (II) or salts thereof can be from entirely different structural classes and have entirely different mechanisms of action. Preference is given to commercially available herbicides as described, for example, in the handbook "The Pesticide Manual", 13th Edition 2003, The British Crop Protection Council, and in "Modern Crop Protection Compounds", edited by Wolfgang Kramer and Ulrich Schirmer, Volume 1 (ISBN 978-3-527-31496-6).

Non-limiting examples of herbicides in relation to which the composition of the present invention may be useful include aminopyralid, glufosinate ammonium, fluroxypyr, imazapic, pendimethlin, sodium chlorate, chloroacetamindes such as metalchlor, acetochlor, butachlor, propachlor, thenylchlor; amides such as dimethenamid, propanil, naptalam, pronamide, bensulide, pethoxamid; organoarsenicals such as cacodylic acid and its sodium salt, disodium methanearsonate, monosodium methanearsonate; benzoic acids and derivatives thereof such as dicamba, chlorfenac, chloramben; nitriles such as dichlobenil and bromoxynil, 2,6-dichlorobenzonitrile, loxynil; benzothiadiazoles such as bentazone; bipyridyliums such as diquat, paraquat, difenzoquat; carbamates such as propham, chloropropham, asulam; phenyl-carbamates such as phenmedipham, desmedipham; chlorinated aliphatic acids such as TCA, dalapon; cyclohexanediones such as sethoxydim, clethodim, cycloxydim, tralkoxydim, tepraloxydim; dinitroanilines such as benefin, oryzalin, pendimethalin, isopropalin; dinitrophenols such as DNOC, DINOSEB; diphenyl ethers such as nitrophen, bifenox, fomesafen, acifluorfen, lactofen, oxyfluorfen; imidazolinones such as imazamox, imazapyr, imazaquin, imazamethabenz-methyl, imazethapyr; triazolopyrimidines such as fiumetsulam, metosulam, chloransulam, disclosulam; aryloxphenoxy propionates such as fluazifop- butyl, propaquizafop, quizalofop-P, metamifop, pyriftalid; phenoxys such as 2,4-D, 2,4,5-T, 2,4-DB, MCPA, silvex, 2,4-DP, MCPB, MCPP; ureas such as fluometuron, linuron, diuron and monuron, fenuron-TCA, siduron, tebuthiuron, isoproturon, cumyluron; phosphono amino acids such as glyphosate, glufosinate, fosamine and glyphosate trimesium; phthalic acid such as chlorthal, endothall; pyridazinones such as pyrazon, norflurazon, fluridone, fluorochloridone, clomazone, oxadiazon, beflubutamid, picolinafen, oxadiargyl; carboxylic acids such as picloram, triclopyr, fluoroxypyr, clopyralid, quinclorac; pyridines such as dithiopyr, thiazopyr; sulfonylureas such as chlorsulfuron, chlorimuron-ethyl, metsulfuron-methyl, sulfometuron-methyl, halosulfuron-methyl, sulfometuron-methyl, bensulfuron, nicosulfuron, triasulfuron, primisulfuron-methyl, amidosulfuron, azimsulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flupyrsulfuron-methyl, foramsulfuron, iodosulfuron-methyl, mesosulfuron-methyl, oxasulfuron, prosulfuron, rimsulfuron, sulfosulfuron, tribenuron-methyl, trifloxysufuron, triflusulfuron-methyl, tritosulfuron, foramsulfuron, iodosulfuron-methyl, thiocarbamates such as EPTC, pebulate, thiobencarb, butylate, cycloate, molinate, vernolate, diallate, triallate; triazines such as atrazine, prometryn, ametryn, and terbutryne; triazinones such as metribuzin and hexazinone; triazoles such as amitrol; uracils such as bromacil, terbacil, lenacil; benzoylcyclohexanediones such as mesotrione, sulcotrione, benzofenap; N-phenyl- phthalimides-cinidon-ethyl; oxazolidinediones such as pentoxazone; phenyl-pyrazoles such as pyraflufen-ethyl; pyrimidindiones such as butafenacil; sulfonyamino-carbonyl-triazolinones such as flucarbazone, propoxycarbazone; tetrazolinones such as fentrazamide; triazolinones such as amicarbazone; flufenpyr-ethyl; oxoziclomefone; benzobicyclon; soaps; petroleum oils. Preferred herbicides which can be applied to the plant in combination with the safener compound of this invention include acetochlor, alachlor, asulam, benfluralin, butachlor, diethatyl, diflufenican, dimethenamid, flamprop, metazachlor, metolachlor, pendimethalin, pretilachlor, propachlor, propanil, trifluralin aminopyralid, chloramben, clopyralid, dicamba, picloram, pyrithiobac, quinclorac, quinmerac, cacodylic acid, copper arsenate, DSMA, MSMA, bensulide, bilanafos, ethephon, fosamine, glufosinate, glyphosate, piperophos, 2,4- D, 2,4-DB, dichlorprop, fenoprop, MCPA, MCPB, 2,4,5-T, dithiopyr, fluroxypyr, imazapyr, thiazopyr, triclopyr, diquat, MPP, paraquat, ametryn, atrazine, cyanazine, hexazinone, prometon, prometryn, propazine, simazine, simetryn, terbuthylazine, terbutryn, chlortoluron, DCMU, metsulfuron-methyl, monolinuron, tebuthiuron, 3-AT, aminocyclopyrachlor, bromoxynil, clomazone, DCBN, dinoseb, mesotrione, methazole, metham sodium, and sulfentrazone.

Yet another group of preferred herbicides to be used in accordance with this invention include, but are not limited to, ACCase inhibitors such as quizalofop, diclofop, fluazifop, fenoxaprop, clethodim, sethoxydim, and pinoxaden; ALS inhibitors such as imazamox, imazapic, imazethapyr, imazaquin, nicosulfuron, metsulfuron, triasulfuron, iodosulfuron, primisulfuron, chloriumuron, tribenuron, chlorsulfuron, thifensulfuron, sulfosulfuron, foramsulfuron, mesosulfuron, prosulfuron, halosulfuron, rimsulfuron, cloransulam-methyl, pyroxsulam, flumetsulam, diclosulam, pyrithiobac, flucarbazone, and propoxycarbazone; root growth inhibitors such as trifluralin, pendimethalin, and ethafluralin; growth regulators such as 2,4-D, 2,4-DB, MCPA, dicamba, clopyralid, fluroxypyr, picloram, and quinclorac; photosynthesis inhibitors (photosystem II) such as atrazine, simazine, prometryn, metribuzin, hexazinone, terbacil, bromoxynil, bentazon, linuron, and diuron; shoot growth inhibitors such as EPTC, metolachlor, alachlor, acetochlor, dimethenamid-P, and flufenacet; aromatic amino acid synthesis inhibitors such as glyphosate; glutamine synthesis inhibitors such as glufosinate; pigment synthesis inhibitors such as norflurazon, clomazone, mesotrione, tembotrione, topramezone, and isoxaflutole; PPO inhibitors such as acifluorfen, fomesafen, lactofen, oxyfluorfen, flumioxazin, flumiclorac, fluthiacet, carfentrazone, and sulfentrazone; photosynthesis inhibitors (photosystem I) such as paraquat and diquat.

Another aspect of the invention is an agrochemical composition comprising a compound of Formula (I) or (II) or a salt thereof as defined hereinabove.

A further aspect of the invention is a plant safener composition comprising the compound of the invention. The composition preferably comprises a compound of formula I at a concentration of 1 μΜ to 100 mM, preferably from 10 μΜ to 10 mM, more preferably from 100 μΜ to 1 mM, e.g. 500 μΜ to 1 mM. The amount of the compound to be applied per plant typically ranges from 1 pmol to 100 mmol, preferably from 10 pmol to 10 mmol, most preferably from 50 pmol to 1 mmol.

The agrochemical composition or the plant safener composition of the invention may further comprise a herbicide. The safener compound of the present invention may be combined with one or more of the herbicides listed above. The safener compounds described herein, together with agrochemical active compounds, preferably herbicides, are suitable for the selective control of weeds in a number of plant crops, for example in crops of economic importance, such as cereals (wheat, barley, rye, triticale, rice, corn, millet), sugar beet, sugar cane, oilseed rape, cotton and soybeans. Of particular interest is the use in monocotyledonous crops, such as cereals (wheat, barley, rye, triticale, sorghum), including corn and rice, and monocotyledonous vegetable crops, but also in dicotyledonous crops, such as, for example, soybean, oilseed rape, cotton, grape vines, vegetable plants, fruit plants and ornamental plants. The present invention can further be used on the following plants as non-limiting examples: tomatoes, squash, pumpkin, beans, broccoli, green beans, asparagus, peas, corn, carrots, spinach, cauliflower, lima beans, broad beans, firench beans, runner beans, navy beans, kidney beans, lentils, cabbage, onions, courgettes, aubergines, sweet basil, leeks, artichokes, lettuce, cassava leaves, tomatoes, cucumbers and gherkins, marrows, gourds, squashes, chilies and peppers, green onions, dry onions, red onions, shallots, garlic, chives, other alliaceous vegetables, okra, mushrooms, watermelons, cantaloupe melons, other melons, bamboo shoots, beets, chards, capers, cardoons, celery, chervil, cress, fennel, horseradish, marjoram, oyster plant, parsley, parsnips, potato, radish, rhubarb, rutabaga, savory, scorzonera, sorrel, sprouts, swede, turnip, watercress and other vegetables, Maize, wheat, rye, oat, triticale, rice, barley, sorghum, millet, buckwheat, fonio, quinoa, spelt, other cereal crops, soybean, peanut, cotton, oilseed rape, sugar cane, bamboo, sesame, jute, canola, coconut, manihot, sunflower, tobacco, ground nuts, peanuts, oil palm, hemp, flax, lucerne, alfalfa tea, perennial grass. Particularly, but not exclusively, when the present invention is used in combination with a herbicide crops of useful plants in which the composition according to the invention can be used include cereals, for example barley and wheat, cotton, oilseed rape, maize, rice, soy beans, sugar beet and sugar cane, especially cereals and maize. Crops can also include trees, such as palm trees, coconut trees or other nuts, and vines such as grapes.

The grasses and weeds to be controlled may be both monocotyledonous species, for example Agrostis, Alopecurus, Avena, Bromus, Cyperus, Digitaria, Echinochloa, Lolium, Monochoria, Rottboellia, Sagittaria, Scirpus, Setaria, Sida and Sorghum, and dicotyledonous species, for example Abutilon, Amaranthus, Chenopodium, Chrysanthemum, Galium, Ipomoea, Nasturtium, Sinapis, Solanum, Stellaria, Veronica, Viola and Xanthium.

The compounds of the formula (I) (or (II) or salts thereof and their combinations with one or more of the abovementioned pesticides can be formulated in various ways, depending on the prevailing physicochemical and biological parameters. Examples of suitable formulation types are known to the person skilled in the art and described, for example, in: K. Martens, "Spray Drying Handbook", 3rd Ed., G. Goodwin Ltd., London, 1979; W. van Valkenburg, "Pesticide Formulations", Marcel Dekker, N.Y. 1973; Winnacker-Kuchler, "Chemische Technologie" [Chemical Technology], volume 7, C. Hanser Verlag Munich, 4th edition 1986; "Perry's Chemical Engineer's Handbook", 5th Ed., McGraw-Hill, N.Y. 1973, pages 8-57. The formulation auxiliaries required, such as inert materials, surfactants, solvents and other additives are also known and are described, for example, in: McCutcheon's "Detergents and Emulsifiers Annual", MC Publ. Corp., Ridgewood N.J.; C. Marsden, "Solvents Guide", 2nd Ed., Interscience, N.Y. 1963; H. von Olphen, "Introduction to Clay Colloid Chemistry", 2nd Ed., J. Wiley & Sons, N.Y.; Sch5nfeldt, "Grenzflachenaktive Athylenoxidaddukte" [Surface- active ethylene oxide adducts], Wiss. Verlagsgesellschaft, Stuttgart 1976; Sisley and Wood, "Encyclopedia of Surface Active Agents", Chem. Publ. Co. Inc., N.Y. 1964; Watkins, "Handbook of Insecticide Dust Diluents and Carriers", 2nd Ed., Darland Books, Caldwell N.J.; Winnacker-Kiichler, "Chemische Technologie", volume 7, C. Hanser Verlag Munich, 4th edition 1986. In addition to the abovementioned formulation auxiliaries, the useful-plant- protecting compositions may comprise, if appropriate, customary tackifiers, wetting agents, dispersants, penetrants, emulsifiers, preservatives, antifreeze agents, fillers, carriers, colorants, anti-foams, evaporation inhibitors and pH or viscosity regulators.

Depending on the formulation type, the useful-plant-protecting compositions generally comprise 0.1 to 99% by weight, in particular 0.2 to 95% by weight, of one or more safeners of the formula (I) or (II), or a combination of safener and pesticide. Furthermore, they comprise 1 to 99.9, in particular 4 to 99.5, % by weight of one or more solid or liquid additives and 0 to 25, in particular 0.1 to 25, % by weight of a surfactant. In emulsifiable concentrates, the concentration of active compound, i.e. the concentration of safener and/or pesticide, is generally 1 to 90, in particular 5 to 80, % by weight. Dusts usually comprise 1 to 30, preferably 5 to 20, % by weight of active compound. In wettable powders, the concentration of active compound is generally 10 to 90% by weight. In water-dispersible granules, the content of active compound is, for example, between 1 and 95% by weight, preferably between 10 and 80% by weight.

For use, the formulations may, if appropriate, be diluted in a customary manner, for example in the case of wettable powders, emulsifiable concentrates, dispersions and water- dispersible granules, with water. Preparations in the form of dusts, granules and sprayable solutions are usually not diluted with any further inert substances prior to use. The required application rate of the safeners varies with the external conditions such as, inter alia, temperature, humidity and the type of herbicide used.

The present invention further provides a method of identifying a receptor molecule binding to a compound of Formula (I) or (II) as defined herein, comprising (i) attaching the compound of Formula (I) or (II) or a salt thereof to a solid support to obtain a modified solid support, (ii) contacting the modified solid support with homogenized plant material, (iii) identifying and optionally characterizing plant molecules bound to the modified solid support. Methods of attaching chemical compounds to suitable solid supports, as well as methods of homogenizing plant material are known to the person skilled in the art. Step (iii) of the method typically comprises washing the solid support (e.g. the column material) to remove unbound plant material not bound to the solid support, and eluting the bound plant material. the eluted compound can be detected by HPLC or other means of detection. The structure of the compounds may then be determined by methods known per se, e.g. by mass spectroscopy, or gas chromatography.

The present invention further provides a method of screening for plant growth-promoting compounds and/or plant safeners, comprising providing a derivative of a compound of Formula (I) or (I I) as defined herein, and determining the plant growth promoting activity and/or the stress tolerance conferring activity of the derivative.

The examples below illustrate the invention and are not to be understood to limit it.

Examples

Example 1 : Plants growing in agar plates

Surface sterilized seeds of Arabidopsis thaliana and Lycopersicum esculentum and were sown on Agar platesl supplemented with 200 μΜ pABA. Six days after germination, plants bear longer root hairs, after 10 days, they exhibited more adventitious roots and more lateral roots than untreated plants (Fig. 1 ). They were 3 replicates for each experiment.

Example 2. Plants growing in soil and watered or sprayed with a solution of pABA

The ability of pABA to promote plant growth when given as a foliar treatment (Arabidopsis thaliana) or incorporated in soil was demonstrated (Arabidopsis thaliana and Zea mays).

Arabidopsis thaliana

Arabidopsis plants were grown from seeds in pots in a mixture of soil and vermiculite and watered with tap water for 15 days. Then watering was stopped for 48h until soil looked dried at the surface of the pot. A total of 4 trails containing 51 pots each were divided into 2 parts. Two trails were watered with deionized water² or deionized water supplemented with 1 mM pABA. The other trails were sprayed with solutions described above. Results show that watering plants with 1 mM pABA induced fresh weight of Arabidopsis aerial parts (leaves ad inflorescence stems) on average of 3.84%%. But when plant were given a foliar application, we observed 17.54% greater fresh weight than plant sprayed with only water³ (Table 1 , Fig. 2). Table 1. Plants growing in soil and watered or sprayed with a solution containing pABA

pABA

Control (n=51 ) (n=51 )

062 ±

Sprayed 0,52 ± 0,02 0,02*

1 ,08 ±

Watered 1 ,00 ± 0,03 0,03

Asterisk (*) indicates significant difference t>95%.

Zea mays

Maize seeds were germinated in Petri® dishes containing deionized-water-humidified Whatman® paper. Germinations of equal size were transferred in pots (one per pot) containing a mixture of soil and vermiculite and watered with tap water for 30 days. Then watering was stopped for 48h until soil looked dried at the surface of the pot. A total of 2 trails containing 10 pots each were respectively were watered with deionized water² or deionized water supplemented with 1 mM pABA.

Results show that watering plants with 1 mM pABA significantly induced fresh weight of maize shoot (Fig. 3, Table 2).

Table 2. Plants growing in soil and watered with a solution containing pABA

Control (n=10) pABA (n=10)

Number of leaves 6.25±0.13 6.58 ±0.14

Root weight (g) 6.39±0.63 7.85±0.49

Shoot weight (g) 4.70±040 6.18*±0.35

Asterisk (*) indicates significant difference t>95%.

Example 3. Induction of adventitious roots on poplar explants

Since pABA can stimulate adventitious development in Arabidopsis, we tested whether pABA could also promotes adventitious development on poplar, a commercially important tree. Poplar explants were grown at the surface of Agar plates¹ , with poplar stems being covered with an Agar plug¹ containing 200 μΜ pABA, or in tubes containing solid medium⁴ supplemented with 200 μΜ pABA prior Agar polymerization. In both plates and tubes, pABA promoted root biomass by inducing an important root system (129% more adventitious than control) (Table 3). Table 3. Activity of pABA of poplar explant.

Control (n=16) pABA (n=20)

Number of leaves 8.68±0.80 8.09±0.59

Adventitious root number 1 .31 ±0.1 1 6.15*(129%)±0.47

Entire plant fresh weight(mg) 203.43±22.40 216±23.69

Roots fresh weight (mg) 42.93±3 68.68* (46%)±6.73

Asterisk (*) indicates significant difference t>95%.

References

Hossain T et al. 2004 Proc. Natl. Acad. Sci. 101 : 5158.

Sahr et al. 2006 Biochem. J. 396: 157-162.

Foot notes

1Arabidopsis growth medium (1/2 MS medium (Murashige and Skoog, 1962).

2deionized water + 1 mM pABA + 1 g L^"1 MES sodium salt, pH 6.9

3deionized water + 1g L^"1 MES sodium salt, pH 6.9

⁴Plants were micro propagated in vitro and grown on 1/2 MS medium (Murashige and Skoog, 962) in glass culture tubes under a 16 h photoperiod at 24°C in a growth chamber.

Example 4

Material and methods

Plant material

adcs T-DNA mutants {SALK_095283 and SALK_034215) were in Arabidopsis thaliana (L.) Heynh., ecotype Columbia (Col-0) background and ugt75b (AY027263) in Landsberg erecta (Ler).

Plant media and treatments

Seeds were surface-sterilized and sown on solid Arabidopsis medium (AM) (2.3 g/liter MS salts, 1 % sucrose, 5mM MES, 1.3% agar-agar (pH 6.0). After vernalization for 2 days at 4 °C, seeds were germinated under long-day period (16 h light, 8 h darkness) at 22 °C. pABA, and 5-FTHF (Folinic acid calcium salt) were dissolved in water used as indicated in result section. B-estradiol was dissolved in 100% ETOH and added to AM without exceeding an ETOH concentration of 0,1 %. Image-capture of plants and growth measurement were performed on 6-day old seedlings, otherwise stated. Construction of plasmids

Plasmids and the conducted modifications are listed in Table S1. Plasmids were constructed according to [36], Gateway^® cloning procedures (Invitrogen), E. coli TOPO10 cells (Invitrogen) was used as a host for constructed plasmids. All genomic sequences were amplified from WT Col-0, GAT-ADCScONA was obtained from RIKEN. GAT-ADCS and HPPK-DHPS related inserts were cloned into the pDONR207 vector while UGT75B related fragments were cloned into pENTR vector. All entry clones were verified through sequencing. Vectors developed by Curtis et al.[26] were used as expression vectors. We developed a novel inducible RNAi vector based on the pMDC7 vector. The gateway cassette in pMDC7 was replaced by the RNAi gateway cassette of pJawohl8 using the restriction sites Spel and X/?o/[37]. All expression clones were generated using the LR-cloning technique (Invitrogen), except pUGT75B::NLS3xYFP [38] which was cloned into the pMDC32 by Hindlll and Sacl after replacing the 35Spromoter and the gateway cassette.

Plant transformation

The agrobacterium strain GV3101 (pMP90) was used to transform Arabidopsis according to the standard floral dip method [39]. Transformants were selected on 15pg/ml Hyromycin B. Quantification of free pABA

Free and total pABA determinations were essentially performed according to [40]. Briefly, seedlings (0.2g to 0.5g) were grinded in liquid nitrogen and extracted two times by 2 ml of methanol. In separate control experiments we verified that the amount of pABA removed by the second extraction represented less than 20% of the pABA removed by the first one, indicating that the yield of extraction was over80%. Both extracts were combined then centrifuged and the supernatant was dried under a nitrogen gas flow. A 1 -ml aliquot of water was added to the residue, followed by sonication for 5 min. The sample solution was then divided into two equal parts of 0.45 ml each, one for the free and the other for the total pABA analyses. For the total pABA analysis, 50 pi of 1 M HCI was added and the tube was incubated at 80°C for 2 hours. After cooling down, 50 μΙ of 1 M NaOH was added for neutralization. Finally, the sample solutions were ultrafiltrated by centrifugation before analysis by reverse phase LC-MS (column: Macherey-Nagel Nucleoshell® RP 18 100x3 mm; HPLC: Agilent Technologies 100 series; mass spectrometer: LTQ from ThermoFisher Scientific). pABA was identified from its retention time and by the MS2 signal corresponding to decarboxylated pABA (precursor ion m/z = 138; product ion m/z = 94). pABA was quantified from standard calibration curves obtained in similar conditions with commercial pABA.

Complementation of adcs2

Self-pollinated heterozygous adcs2 were transformed with the genomic fragment of GAT- ADCS (-2175bp to 4364bp). Transformants were confirmed as adcs2 mutants by PCR and the ratio of defective embryos was determined. Complemented single insertion lines were expected to display a ratio of 6.25% defective embryos, while for double insertion lines a ratio of 1.56% was expected.

Plant scanning and microscopy

Seedlings were imaged while growing in plate with a flatbed CanonScan 9950F scanner. Histological detection of β-glucuronidase (GUS) activity and plant preparation for microcopy were performed according to [41]. For light microscopy samples were observed with a Zeiss Axiovert 200M MOT (Carl Zeiss Microimaging) for high magnification pictures. Low magnification views were taken with a Zeiss Stemi SV1 1 Apo stereomicroscope (Carl Zeiss Microimaging), viewed under differential interference contrast (DIC) optics. Fluorescent proteins were analyzed with a Zeiss LSM 5 DUO scanning microscope and AZ-C1 Macro Laser Confocal Microscope from Nikon. To monitor GFP and DAPI fluorescence, we used multitracking in frame mode. GFP was excited using the 488 nm laser line in conjunction with a 505-530 band-pass filter. DAPI was excited with the 405 nm laser line and collected using a 420-480 nm band-pass filter. For VENUS imaging, proteins were excited using the 514 nm laser line, and the emission collected at 527 nm. Images were analyzed with the LSM image browser (Carl Zeiss Microimaging) and Bitplanelmaris®.

Results

Tissue localization of pABA and folate syntheses in Arabidopsis thaliana

First we visualized the sites of pABA and folate synthesis in planta. We constructed reporter lines to visualize the expression domains of GAT-ADCS and HPPK-DHPS, the genes required respectively for pABA synthesis[16] and pABA incorporation into the THF pathway [7](Figure 4A). For pABA synthesis, GAT-ADCS promoter was fused to the β-GIucoronidase (GUS) reporter gene [17]. GAT-ADCS was expressed in pollen grains and during female gametophyte ontogenesis (Figure 5). During embryo development, GAT-ADCS was expressed predominantly in endosperm until the transition globular-heart stage. Thereafter, GAT-ADCS expression dropped in maternal tissues and was shifted to developing embryo (Figure 5). In seedlings, GAT-ADCS promoter was active throughout the plant (Figure 4C). In shoots, GUS signal was present in cotyledons and leaves mainly in vasculature and stomata and in shoot apical meristem. GAT-ADCS expression was detected at root-shoot junction and along root vasculature up to the elongation zone (Figures 4C and 12A). At root tip, for short time GUS-staining (30 min), GAT-ADCS was expressed in the QC and in columella lateral root cap cells (Figure 4D, left). When plants were stained for longer time (3h), pADCS::GUS signal could be detected in epidermis and cortex (Figure 4D, middle). HPPK- DHPS catalyzes the fusion of pABA and pterin for folate synthesis [7]. HPPK-DHPS promoter activity globally matched GAT-ADCS promoter expression pattern in the whole plant (Figure 4C and 4D) strongly suggesting that pABA is produced at the site of its utilization for THF synthesis.

GAT-ADCS is the key enzyme for plant development and pABA synthesis in Arabidopsis thaliana

The expression pattern of GAT-ADCS during embryo development suggested that pABA might be involved in early steps of plant ontogenesis. To confirm the importance of pABA synthesis for plant development, we isolated two lines carrying T-DNA insertions in the GAT- ADCS gene in the Columbia (Col-0) background which we named adcs-1 (SALK_034215) and adcs-2 (SALK_095283). adcs-1 has a T-DNA inserted 48bp upstream of the transcriptional starting point (Figure 6A). Surprisingly homozygous adcsl showed no growth alterations suggesting that the remaining portion of the promoter was still active (Figure 7). However, adcs-2 which has a T-DNA inserted in the 12^th exon produced 25% embryos arrested at the globular stage (Figures 6A-D). These embryos could develop further when opened siliques were transplanted on a medium supplemented with pABA, confirming these mutants lacked synthesis of pABA dependent metabolites (Figure 8A-C). In support of the latter conclusion, shoots not in contact with the medium did not develop further (Figure 8 D). Moreover, adcs-2 mutants could be complemented by GAT-ADCSYJT allele, reducing the ratio of arrested embryos from 25% to 6.25%, the expected ratio for a complemented heterozygous mutant (Figure 6F and 6G, lines 10 and 17). Some complemented lines displayed an embryo defective ratio of 1.56%, indicative of a double insertion of the complementing construct (Figure 6F and 6G, line 1 1 ). The observation that roots of adcs-2 could grow further on pABA supplemented medium suggests that pABA was indeed absorbed by the roots and metabolized. All together these results indicate the existence of an active pathway for pABA biosynthesis in roots. To further connect pABA requirements and folate biosynthesis, we engineered a β-estradiol (EST) inducible RNAi targeting a fragment of GAT-ADCS encoding for a GAT-ADCS specific linker region [18].

When applied to roots, EST induced down regulation of GAT-ADCS expression which resulted in a root growth arrest, again supporting the importance of de novo pABA synthesis for root growth (Figure 9A and 9B). As expected, arrested roots could be rescued by adding either pABA or a folate derivative (5-formyl-tetrahydrofolate, 5-FTHF, a stable derivative that is readily converted to metabolically active folates once incorporated in cells)(Figure 9C). Taken together these results demonstrate unequivocally that GAT-ADCS-dependent de novo synthesis of pABA in root tips is required for folate synthesis and thereby for root growth. pABA displays an innate folate-independent activity on root growth

To get more insights into the pABA role on plant development, plants were grown in presence of pABA. Concentrations of pABA greater than 50μΜ inhibited root growth and promoted lateral and adventitious roots development as well as root hair growth without affecting the aerial part of the plant (Figure 10A--E). In contrast, 5-FTHF applied at similar concentrations did not phenocopy the activity of pABA (Figure 10B). To verify if both compounds were taken up by roots, we incubated plants with pABA and 5-FTHF respectively in association with sulfanilamide (SULF), a pABA antagonist used as an inhibitor of folate synthesis [19]. In both cases SULF inhibition on plant development was reverted, confirming that pABA and 5-FTHF were indeed absorbed and metabolized (Figure 10F).

Moreover, besides its activity on root elongation, pABA, but not 5-FTHF, also suppressed plant-root waving (Figure 10B). Arabidopsis root waving occurs when plants are grown at the surface of an agar plate that is slightly tilted. This growth response is strongly dependent on positive gravitropism[20]. To test whether pABA modulates root response to gravity, agar plates bearing Arabidopsis seedlings were rotated with an angle of 135^° and their graviresponse was quantified (Figure 10G). For AM and 5-FTHF treatments, three hours after gravistimulation, the majority of root tips displayed angles with the vertical axis comprised between 60 and 120^° (with 0^° representing full recovery). For pABA-treated seedlings, root tips showed enhanced curvature, with the majority of angles between 30° and 90°, indicating that roots responded faster to gravistimulation. Taken together these data show that pABA and 5-FTHF have different activities and therefore suggest that pABA possesses an innate folates-independent activity on plant growth. Regulation of pABA activity through conjugation of pABA to glucose

pABA exists under two forms: an unconjugated metabolically active form presumably located in the cytoplasm (cytosol plus organelles), and an inactive conjugated form located in the vacuole (Figures 4A and 11 A). In control plants, free pABA is very low representing 5% of total pABA, in average. This low concentration (probably less than 1 μΜ in the cytoplasm, assuming a cytoplasmic volume representing 10 % of the cell volume) makes accurate determination of free pABA quite difficult because, as previously shown QO], breakdown of the esterified form of folate during sample workup is very difficult to avoid and contributes, sometimes quite significantly, to the amount of free pABA.

In order to understand how pABA homeostasis is controlled, we examined the role of this conjugation process on root growth and development. In plants pABA is preferentially conjugated to glucose by the pABA-glucosyltransferaseUGT75B (AT1 G05560) [1 1]. ^Tnis enzyme is active throughout the plant including leaves, flowers, siliques, and root [1 1]. To know precisely in which tissues pABA is conjugated to glucose, we investigated the expression pattern of the glucosyltransferase encoding UGT75B gene. The promoter of UGT75B was fused to a nuclear targeted YFP (NLS3xYFP) [21]. The expression pattern in the root tip was similar to those of GAT-ADCS and HPPK-DHPS. Like GAT-ADCS, UGT75B was expressed in lateral root cap cells (LRC), in epidermis and cortex (Figure 12).

To determine how the regulation of pABA pools may influence pABA activity on root development, we analyzed a ugt75b knock-out mutant and then generated stable conditional lines differentially overexpressing UGT75B. As previously shown [9], knocking out expression of the UGT75B gene resulted in a 3-4 fold decreased of the pABA-glucose level and a 5 time increase of the free form (Figure 1 1 B). The roots of the ugt75b mutant displayed higher sensitivity to pABA than wild type (Figure 13). At low concentration of pABA (10μΜ), 60% percent of ugt75B seedlings showed emerged adventitious roots (AR) versus only 20% for wild-type (WT) plants (Figure 13A). Similarly, root growth of ugt75b appeared more sensitive to pABA and was already significantly inhibited at 30 μΜ (Figure 13B).

Conditional induction of UGT75B (Lex::UGT75B) with 1 μΜ β-estradiol (EST) resulted in a two fold increase of pABA-glucose compared with WT (Figure 1 D). Note that the non- induced UGT75B (Figure 1 1 C) also displayed an increased level of pABA- glucose (about 1.5 fold the WT) indicating some overexpression of UGT75B even in the absence of EST. Due to the low initial level of free pABA, it was not possible in these experiments to determine any significant change within the free pABA pool. However, taking into account that the increase of pABA-glucose resulting from the overexpression of UGT75B required the contribution of the free pool, this pool might be less available for other metabolic purposes. From this point of view, conditional induction of UGT75B with high concentration of EST (20μΜ), abolished root growth and induced exaggerated root waving (Figures 13D and 14). Upon weak β-estradiol induction (0.1 μΜ), root growth was retarded and roots displayed a right-hand slanting (Figure 13E). These two phenotypes could be suppressed by exogenous pABA when only applied at high concentration (from 200μΜ). In addition, rescued plants became slightly resistant to pABA and developed longer roots than pABA-treated wild type (Figure 13E and G). Furthermore, since the glucosylation of pABA could have dampened the biosynthesis of folates, we checked whether 5-FTHF could suppress observed growth defects. 5-FTHF supplementation had no effect on β-estradiol-induced UGT75B plants (Figure 13E-G), indicating that growth alterations were not due to folate deficiency. Upon strong UGT75B induction, root growth was only partially recovered in the presence of pABA and very poorly in the presence of 5-FTHF (Figure 14A and 14B). pABA impacts on processes responsible for auxin distribution in root epidermis

Plant growth relies on auxin gradients maintained by series of auxin influx carriers of the AUXIN RESISTANT 1/ Like AUX1 (AUX/LAX) family [22], efflux facilitators such as ABCB/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) [23], and PIN-FORMED (PIN) proteins [24]. PIN-proteins subcellular localization is highly dynamic and influenced by various internal and external signals, thereby directing and redirecting the intercellular fluxes of auxin[25J. Hence, the polarity of PIN-proteins is for example used to indicate the direction of auxin flux [26]. During plant response to gravity, auxin transport through AtPIN2 plays a crucial role and pin2 mutants are unable to establish lateral auxin gradient after gravi- stimulation and therefore are agravitropic [27,28]. Plants treated with pABA were hypergravitropic (Figure 10D and 10G), while induced Lex::UGT75B plants showed disturbed root graviresponse (Figures 13D and 15A) suggesting that pABA could play a role in modulating the activity of some components of the auxin signaling pathway important for root graviresponse. To test this hypothesis we visualized both PIN1 and PIN2 localization and auxin gradient in roots tips of plants treated with exogenous pABA or deprived of free endogenous pABA by overexpressing UGT75B. In WT and Lex::UGT75B (non induced) root tips treated or not with pABA, PIN1 and PIN2 displayed their typical localization pattern. In the stele up to cortex cells, PIN1 was basally (rootward) localized. PIN2 was localized apically (shootward) in epidermis channeling auxin basipetally, and rootward in cortex cells (from root-transition zone) transporting auxin back to the QC (Figure 15B). Epidermis- shootward and cortex- rootward PIN2 auxin transport reflux loop consolidate root-tip auxin maxima (as measured by the auxin-sensitive DR5 promoter), which is important for cell division and growth (Figures 15B and 16A) [29]. However, while estradiol-treated Lex::UGT75B roots displayed no change for PIN1 subcellular localization, PIN2 polarity was lost in both epidermis and cortex cells. PIN2 was found at plasma membrane around the cell and in the cytoplasm of 80% (n=10) of plants analyzed (Figure 15B). As a consequence, DR5::GUS signal also was perturbed in 100% (n=10) of plants analyzed (Figure 16D). Again, PIN2 polarity and root response to gravity of induced Lex::UGTB75B could be restored by pABA (250μΜ), but not by 5-FTHF at similar or even higher concentrations (up to 1 mM) (Figure 15A and 15B).

Example 5: Effects of Benzoic acid and its derivatives on root hairs

It was tested whether benzoic acid derivatives can counteract the activity of natural auxins (IAA) (Figure 17 and Figure 18). The experiments were carried out as described for Figure 10. Briefly, Arabidopsis seedlings (Wild type Col 0) were cultivated on (a) control medium (AM) and AM supplemented with (b) 100μΜ pABA, (c) 1 μΜ IAA or (d) 1 μΜ IAA + 100μΜ pABA (Figure 17, upper panel). As can be seen from Figure 17, pABA could antagonize the growth-inhibitory effects of IAA. In a similar experiment, Arabidopsis seedlings (Wild type Col 0) were cultivated on control medium (AM), AM supplemented respectively with 3-ABA, pABA, Procaine, 5-FTHF, applied at the same concentration (100 μΜ), in the absence (Figure 18, white bars) or presence (Figure 18, black bars) of 2 μΜ IAA. Benzoic acid derivatives such as pABA, mABA, Procaine and 5-FTHF fully restored root growth in the presence of inhibory concentrations of IAA.

Further, the activity of benzoic acid derivatives on root growth was studied using Arabidopsis seedlings cultivated on Control (AM) medium (Mock) or AM supplemented with either 100 μΜ pABA, N0₂-BA, CI-BA, l-BA or F-BA. It was found that benzoic acid derivatives having a pK_a value of less than 4 had little or no root growth-promoting effect. On the other hand, pABA which has a pK_a value of about 4.9 had significant root growth-promoting activity (Figure 19).

Example 6: Confirmation of PABA safener activity Goal 1

Test the activity of pABA as safener by analysing the impact of pABA of root growth in absence of or in presence of exogenously applied auxin (indole 3-acetic acid, IAA). Goal 2

Test the activity of pABA as safener by analysing the impact of pABA of root growth in absence of or in presence of the herbicide N-1 -naphthylphthalamic acid (NPA). NPA is a chemical inhibitor of polar auxin transport (PAT). Plant Material

Arabidopsis thaliana seedlings expressing the auxin reporter DR5::GFP. The coding sequence of the green-florescent protein (GFP) is driven by the synthetic auxin responsive DR5 promoter.

Results

Goal 1

Compared to untreated plants, 1 μΜ IAA is toxic for plant growth as it represses root growth (62% inhibition) (Figure 20A-B). Under the same growth conditions, 100μΜ PABA only slightly affected main root growth (8.3% of untreated plants). However when IAA and PABA were simultaneously applied, PABA counteracted the activity of IAA and restored root growth (up to 90% when compared to the root size of untreated plants) (Figure 20A-B), demonstrating that pABA can act as a safener, protecting plants against the toxic activity of IAA on root growth.

Goal 2

NPA applied atl OpM suppresses PAT and is therefore toxic for plant growth. NPA induces 50% root growth-inhibition and strongly perturbs root response to gravity (note plant roots are growing upside down) (Figure 20A-B). When NPA and PABA were simultaneously applied, pABA counteracted the activity of NPA and improved root growth (up to 60% when compared to the root size of untreated plants) and also restored root response to gravity (Figure 20A- B), demonstrating the safener activity of pABA, protecting plants against the toxic activity of NPA.

To further understand how pABA regulates root response in presence of IAA and NPA. Both toxic compounds were simultaneously applied to plants, resulting in the strong inhibition of the overall plant development, with the main root growth inhibited for about 87%. Moreover, inhibited roots were also agravitropic (Figure 20A-B). But when pABA was associated together with IAA and NPA, root growth and root response to gravity were restored confirming the safener activity of pABA (Figure 20A-B).

Furthermore, we visualized the auxin response in control plants (untreated), in plants grown in presence of 100μΜ pABA, in presence of 1 μΜ IAA or 10μΜ NPA, or when these chemicals were combined (as described above). In untreated plants, DR5 signal shows a maximum the root tip in quiescent centre, collumela stem cells and differentiated root cap cells (Figure 20C). In presence of IAA or pABA, both chemical promote an extension of the auxin response in roots extending in lateral root cap cells and vascular cells (Figure 20C) suggesting that pABA probably impacts on auxin pathways in planta. In presence of NPA, DR5::GFP maximum expands to the entire root tip, including epidermis and cortex cell files (Figure 20C). This response reflects the inhibition of auxin transport by NPA. When applied together IAA and PABA show additive effects on DR5::GFP signal suggesting that PABA and IAA activate similar pathways. More strikingly, when combined with NPA or with IAA+NPA, pABA reduced the NPA impact of DR5::GFP signal. All together, these data show that PABA safener activity on toxic NPA effect on root growth probably involves auxin transport.

References

1 . Ravanel S, Douce R, Rebeille F (201 1 ) Metabolism of folates in plants. In: Rebeille F, Douce R, editors. Advances in Botanical Research, Vol 59. Amsterdam, The Netherlands:

Elsevier, pp. 67-106.

2. Hanson AD, Gregory JF (201 ) Folate Biosynthesis, Turnover, and Transport in Plants. In: Merchant SS, Briggs WR, Ort D, editors. Annual Review of Plant Biology, Vol 62. Palo Alto: Annual Reviews, pp. 105-125.

3. Mehrshahi P, Gonzalez-Jorge S, Akhtar TA, Ward JL, Santoyo-Castelazo A, et al. (20 0) Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development. Plant Journal 64: 267-279.

4. Basset G, Quinlivan EP, Ziemak MJ, Diaz de la Garza R, Fischer M, et al. (2002) Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proceedings of the National Academy of Sciences 99: 12489-12494.

5. Camara D, Richefeu-Contesto C, Gambonnet B, Dumas R, Rebeille F (201 1 ) The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana. Arch Biochem Biophys 505: 83-90.

6. Basset GJC, Ravanel S, Quinlivan EP, White R, Giovannoni JJ, et al. (2004) Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant Journal 40: 453-461 .

7. Rebeille F, Macherel D, Mouillon J-M, Garin J, Douce R (1997) Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8- di hydro pterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria. EMBO J 16; 947-957.

8. Ishikawa T, Machida C, Yoshioka Y, Kitano H, Machida Y (2003) The GLOBULAR ARREST1 gene, which is involved in the biosynthesis of folates, is essential for embryogenesis in Arabidopsis thaliana. Plant Journal 33: 235-244.

9. Neuburger M, Rebeille F, Jourdain A, Nakamura S, Douce R (1996) Mitochondria are a major site for folate and thymidylate synthesis in plants. Journal of Biological Chemistry 271 : 9466-9472.

10. Quinlivan EP, Roje S, Basset G, Shachar-Hill Y, Gregory JF, et al. (2003) The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol.

Journal of Biological Chemistry 278: 20731-20737.

1 1. Eudes A, Bozzo GG, Waller JC, Naponelli V, Lim EK, et al. (2008) Metabolism of the folate precursor p-aminobenzoate in plants - Glucose ester formation and vacuolar storage. Journal of Biological Chemistry 283: 15451 -15459.

12. Ludwig-Muller J (201 1 ) Auxin conjugates: their role for plant development and in the evolution of land plants. Journal of Experimental Botany 62: 1757-1773.

13. Sahr T, Ravanel S, Rebeille F (2005) Tetrahydrofolate biosynthesis and distribution in higher plants. Biochemical Society Transactions 33: 758-762.

14. Jabrin S, Ravanel S, Gambonnet B, Douce R, Rebeille F (2003) One-carbon metabolism in plants. Regulation of tetrahydrofolate synthesis during germination and seedling development. Plant Physiology 131 : 1431-1439.

15. Nawy T, Lee JY, Colinas J, Wang JY, Thongrod SC, et al. (2005) Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 17: 1908-1925.

16. Basset GJC, Quinlivan EP, Ravanel S, Rebeille F, Nichols BP, et al. (2004) Folate synthesis in plants: The p-aminobenzoate branch is initiated by a bifunctional PABA-PabB protein that is targeted to plastids. Proceedings of the National Academy of Sciences of the United States of America 101 : 1496-1501.

17. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901 -3907.

18. Hilson P, Allemeersch J, Altmann T, Aubourg S, Avon A, et al. (2004) Versatile gene- specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. Genome Res 14: 2176-2189.

19. Sankar DVS (1958) Para-Amino-Benzoic Acid and Sulphanilamide in the Nutrition of Neurospora-Crassa. Nature 181 : 629-630.

20. Okada K, Shimura Y (1990) Reversible Root-Tip Rotation in Arabidopsis Seedlings Induced by Obstacle-Touching Stimulus. Science 250: 274-276. 21. Sarkar AK, Luijten M, Miyashima S, Lenhard M, Hashimoto T, et al. (2007) Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 446: 81 1 -814.

22. Ugartechea-Chirino Y, Swamp R, Swamp K, Peret B, Whitworth M, et al. (2010) The AUX1 LAX family of auxin influx carriers is required for the establishment of embryonic root cell organization in Arabidopsis thaliana. Annals of Botany (London) 105: 277-289.

23. Geisler M, Murphy AS (2006) The ABC of auxin transport: the role of p-glycoproteins in plant development. FEBS Lett 580: 1094-1 102.

24. Paponov IA, Teale WD, Trebar M, Blilou I, Palme K (2005) The PIN auxin efflux facilitators: evolutionary and functional perspectives. Trends Plant Sci 10: 170-177.

25. Grunewald W, FrimI J (2010) The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells. EMBO J 29: 2700-2714.

26. Wisniewska J, Xu J, Seifertova D, Brewer PB, Ruzicka K, et al. (2006) Polar PIN localization directs auxin flow in plants. Science 312: 883.

27. Muller A, Guan C, Galweiler L, Tanzler P, Huijser P, et al. (1998) AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J 17: 6903-691 1.

28. Ottenschlager I, Wolff P, Wolverton C, Bhalerao RP, Sandberg G, et al. (2003) Gravity- regulated differential auxin transport from columella to lateral root cap cells. Proc Natl Acad Sci U S A 100: 2987-2991.

29. Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, et al. (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature (London) 433: 39-44.

30. Hanson AD, Gregory JF, 3rd (201 1 ) Folate biosynthesis, turnover, and transport in plants. Annu Rev Plant Biol 62: 105-125.

31. Meinke DW, Meinke LK, Showalter TC, Schissel AM, Mueller LA, et al. (2003) A sequence-based map of Arabidopsis genes with mutant phenotypes. Plant Physiology 131 : 409-418.

32. Patton DA, Schetter AL, Franzmann LH, Nelson K, Ward ER, et al. (1998) An embryo- defective mutant of arabidopsis disrupted in the final step of biotin synthesis. Plant Physiol 1 16: 935-946.

33. Srivastava AC, Ramos-Parra PA, Bedair M, Robledo-Hernandez AL, Tang Y, et al. (201 1 ) The folylpolyglutamate synthetase plastidial isoform is required for postembryonic root development in Arabidopsis. Plant Physiol 155: 1237-1251.

34. Kutschera U, Bergfeld R, Schopfer P (1987) Cooperation of Epidermis and Inner Tissues in Auxin-Mediated Growth of Maize Coleoptiles. Planta 170: 168-180. 35. Dhonukshe P, Huang F, Galvan-Ampudia CS, Mahonen AP, Kleine-Vehn J, et al. (2010) Plasma membrane-bound AGC3 kinases phosphorylate PIN auxin carriers at TPRXS(N/S) motifs to direct apical PIN recycling. Development 137: 3245-3255.

36. Sambrook JF, E.F., Maniatis, T., editor (1989) Molecular cloning: a Laboratory Manual. New York.

37. Feys BJ, Wiermer M, Bhat RA, Moisan LJ, Medina-Escobar N, et al. (2005) Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY 1 complex in plant innate immunity. Plant Cell 17: 2601 -2613.

38. Breuninger H, Rikirsch E, Hermann M, Ueda M, Laux T (2008) Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo. Dev Cell 14:

867-876.

39. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743.

40. Camara D, Bisanz C, Barette C, Van Daele J, Human E, et al. (2012) Inhibition of p- Aminobenzoate and Folate Syntheses in Plants and Apicomplexan Parasites by Natural

Product Rubreserine. Journal of Biological Chemistry 287: 22367-22376.

41. Ditengou FA, Teale WD, Kochersperger P, Flittner KA, Kneuper I, et al. (2008) Mechanical induction of lateral root initiation in Arabidopsis thaliana. Proc Natl Acad Sci U S A 105: 18818-18823.

Claims

Claims

1. The use of a compound of Formula (I)

or a salt thereof as a safener,

wherein

n is 0, 1 , 2, 3, 4 or 5,

R¹ is -OR³, -NR⁴R⁵, halogen, or a hydrocarbon chain with 1 to 6 carbon atoms,

each R² is independently selected from the group consisting of amino, nitro, cyano, halogen, (C_rC₆)-alkyl, (C₂-C₆)-alkenyl, (C₂-C₆)-alkinyl, (C C₆)-alkylamino, (C₂-C₆)-alkenylamino, (C₂- C₆)-alkinylamino, (C C₆)-alkoxy, sulfonylamino, (C_rC₆)-alkylsulfonylamino, amino-(C_rC₆)- alkyl, amino-(C₂-C₆)-alkenyl, amino-(C₂-C₆)-alkinyl, hydroxycarbonyl, aminocarbonyl, R³ is hydrogen or optionally substituted (C C₆)-alkyI,

each of R⁴ and R⁵ is independently selected from the group consisting of hydrogen and optionally substituted (C C₆)-alkyl;

characterized in that the pK_a value of the compound is at least 4.0.

2. The use of claim 1 , wherein R¹ is -OH or -NH₂.

3. The use of any one of the preceding claims, wherein n is 0 or 1.

4. The use of any one of the preceding claims, wherein the pK_a value of the compound is at least 4.2.

5. The use of any one of the preceding claims, wherein the pK_a value of the compound is at least 4.6.

6. The use of any one of the preceding claims, wherein the pK_a value of the compound is at least 4.8.

7. The use of any one of the preceding claims, wherein each R² is independently selected from the group consisting of amino, cyano, and methyl.

8. The use of claim 7, wherein each R² is amino.

9. The use of any one of claims 1 to 8, wherein one group R² is attached to the carbon atom at position 3 or 4 of the aromatic ring.

10. The use of claim 9, wherein the compound is 4-Aminobenzoic acid (pABA) or 3- Aminobenzoic acid (mABA).

1 1. The use of a compound of Formula (I) or a salt thereof as defined in any one of claims 1 to 10 as a plant growth promoter.

12. An agrochemical composition comprising a compound of Formula (I) or a salt thereof as defined in any one of claims 1 to 10.

13. The agrochemical composition of claim 2, which is a safener composition.

14. The composition of claim 12 or 13, further comprising a herbicide.

15. A method of promoting the growth of a plant, comprising applying to the plant a compound of Formula (I) as defined in any one of claims 1 to 10.

16. A method of improving the stress tolerance of a plant, comprising applying to the plant a compound of Formula (I) as defined in any one of claims 1 to 10.

17. The method of claim 16, wherein the stress tolerance is tolerance to one or more herbicides.

18. The method of claim 16 or 17, wherein the plant has been treated, is treated or is to be treated with a herbicide.

19. A method of detecting a receptor molecule binding to a compound of Formula (I) as defined in any one of claims 1 to 10, comprising (i) attaching the compound of Formula (I) or a salt thereof to a solid support to obtain a modified solid support, (ii) contacting the modified solid support with homogenized plant material, (iii) identifying and optionally characterizing plant molecules bound to the modified solid support.

20. A method of screening for plant safeners, comprising providing a derivative of a compound of Formula (I), and determining the stress tolerance conferring activity of the derivative.

21. A method of screening for plant growth-promoting compounds, comprising providing a derivative of a compound of Formula (I), and determining the plant growth promoting activity of the derivative.