New Zealand No. International No
320845
TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION
Priority dates 21 091995,
Complete Specification Filed 20 091996
Classification (6) A01H5/00, A01N3/00
Publication date 28 October 1998
Journal No 1433
Title of Invention
Conditionally male-fertile plants and methods and compositions for restoring the fertility thereof
Name, address and nationality of applicant(s) as in international application form
WASHINGTON STATE UNIVERSITY RESEARCH FOUNDATION, N E 1615 Eastgate Boulevard, Pullman, WA 99164-1802, United States of America
NEW ZEALAND PATENTS ACT 1953
COMPLETE SPECIFICATION
3208
WO 97/10703 PCT/US96/15131
CONDITIONALLY MALE-FERTILE PLANTS AND METHODS AND COMPOSITIONS FOR RESTORING THE FERTILITY THEREOF
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority to co-pending U S provisional patent application serial no 60/004,214, filed on September 21, 1995, which is incorporated herein by reference
TECHNICAL FffilP
The present invention is related to male sterility in plants and more parucularly to compositions and methods for producing and/or identifying S conditionally male-fertile plants and for restoring fertility to such conditionally male-fertile plants
PACKQRQVNP ART
The regulation of male fertility in higher plants has tremendous 10 practical importance to the breeding of crop and horticultural plants Dominant, recessive, nuclear, and cytoplasmic male-sterile genetic traits are used in various breeding schemes The most economically-important use of male sterility is in hybrid plant breeding In most higher plant species, hybrid cultivars are generally superior to open-pollinated cultivars in yield or in other 15 production-related characteristics Hybrid plant breeding requires a functionally male-stenle plant as the female parent, but the hybrid must be fully fertile if seeds or fruit are the harvested crop Furthermore, to take full advantage of heterosis, the female parent must come from an inbred line
Two basic approaches have been employed in plant breeding in order 20 to balance these requirements In the first approach, a male-fertile line is employed during inbreeding, then the inbred line is rendered male-stenle during the production phase (i.e , the step in which the cross is made to the male parental line to produce seed) Mechanical detasseling of corn, the use of male gametocides in wheat, and hand emasculation in tomato are examples of this
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approach A drawback to this first approach is the necessity of handling the large numbers of flowers involved in the production phase
In the second approach, a cytoplasmic male-stenle line and a normal-cytoplasm mamtainer (fertile analagon line) are used during in-breeding 5 of the female parent The male parental line in the breeding program contains a fertility-restoring allele (e g , of a nuclear gene, Rf) that overcomes the cytoplasmic malc-sterility trait to ensure that the hybrid is fertile A breeding scheme employing a cytoplasmic male-sterile parental line is complex, requiring identification of both a suitable cytoplasmic male-sterile trait and a suitable 10 dominant fertility-restoring allele The cytoplasmic male-sterile and mamtainer lines must be earned together during the inbreeding phase
Nuclear-encoded male-stenle traits have not been widely used in hybnd breeding systems In principle, recessive male-stenle traits can be maintained as hetercrvgotes during inbreeding with bomozygous plants being IS chosen for crosses wiui the male parental line Several strategies have been developed to assist this approach, but the difficulty of obtaining homozygous male-stenle plants for the production phase has limited the use of this approach in practice These strategies are desenbed in greater detail, eg., in Kaul, Male Sterility :n Higher Plants, Springer-Verlag, Berlin, 1988, and U S Patents 20 4,654,465 and 4,727,219. which are incorporated herein by reference
Several cntena are important for practical application of these approaches in hybnd breeding programs, including the following
1 Dunng the production phase there is a more or less absolute requirement for functional male-stenlity Self-pollination of the female parent 25 compromises the puntv of hybnd seed produced This consideration is particularly important for species, such as com, for which the inbred lines used show strong inbreeding depression and the heterosis observed m the hybnds is high In other crops in which inbreeding depression and heterosis are modest, a low level of non-hybnd seed may be acceptable Ensunng male-stenlity in the 30 female parent requires careful control of emasculation techniques such as detasselmg or the use of male gametocides When genetic male-steriiity is employed, the female parent must be consistently male-sterile under all the
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environmental conditions (and in all genetic backgrounds) used in the production phase
2 The emasculation treatment or genotype must have no significant effect on female fertility, since this would compromise hybrid seed production
In addition, the female parent used must be free of any barriers to cross-pollination by the male parent
3 Similarly, the emasculation treatment or genotype must not significantly reduce the growth and productivity of the hybnd plants A cytoplasmic male-sterile trait must be completely recessive in the presence of the
restorer and preferably does not confer an undesirable phenotype such as susceptibility to a plant pathogen A nuclear male-stenle trait must also be completely recessive and free of undesirable pleiotropic effects
It would be highly advantageous to use a recessive nuclear male-stenle trait as a hybnd breeding tool This would be possible if the line 15 could be maintained as a Homozygous male-stenle line and rendered conditionally fertile during each generation of the inbreeding phase Such an approach could provide the large numbers of uniformly male-stenle plants needed during the production phase while requiring restoration of fertility in the small numbers of plants handled dunng each inbreeding cycle 20 There have been attempts to produce condiuonal male fertility by the administration of exogenous chemicals, including flavonoid compounds (Taylor et al , J Hered 83 11-17, 1992, WO 93/18142) and plant growth regulators, including gibberellic acid (Kaul, Male Sterility in Higher Plants,
Springer-Verlag, Berlin, Heidelberg, pp 15-96, 1988) In general, however, 25 these technologies are not widely used in the production of hybrid plants
A complex system for the establishment of conditional male fertility involves incorporating into the genome of a plant a transgene the expression of which can be induced by administration of a chemical inducer The transgenic plant is normally male sterile but is rendered male-fertile upon expression of the 30 transgene Such a system is desenbed in U S Patent No 5,432,068
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PPIEF DESCRIPTION QF THE PRAWTNgS
FIG 1 shows the relative growth rate of wild-type Arabidopsis and the fad3 fad7-2 fad8 triple mutant At intervals between 8 and 24 days after sowing, samples of plants were harvested and the fresh weight of the above-5 ground parts was measured The relative growth rate (oj ') for the wild-type (•) was 0 388 ± 0 025 and that for the mutant (o) was 0 388 ± 0 033
SUMMARY OF THE INVENTION
It has been discovered that the fertility of certain male-strnle plants 10 can be restored by the application of exogenous jasmonate or a related compound to the plants These male-stenle plants are referred to herein as "conditionally male-fertile" plants
The conditionally male-fertile phenotype is caused by mutations that interfere with normal jasmomc acid metabolism Such mutations can include, but IS are not 1 united to, mutations that interfere with biosynthesis of jasmomc acid from a-linolenate (e g , mutations in structural genes for enzymes in the jasmonate biosynthetic pathway) or trans-acting regulators of such structural genes or their binding sites, receptors for jasmonate or its precursors; etc
The present invention is useful for identifying conditionally male-20 fertile plants arising naturally or as a result of various conventional mutagenesis techniques, including genetic engineering This discovery also facilitates the production of additional conditionally male-fertile plant varieties for use, for example, in hybnd breeding programs The ability to produce and/or identify conditionally male-fertile plants and to rescue the male-fertility of these plants by 25 simply applying a chemical compound forms the basis for a conditional male-fertility system that is broadly applicable to hybnd breeding of crop and horticultural plants
Therefore, one aspect of the present invention encompasses compositions that include a conditionally male-fertile plant and an effective 30 amount of jasmonate or a related compound, i e , an amount of a composition comprising jasmonate or a related compound that is effective to restore male fertility to the plant when applied to the plant, preferably jasmomc acid, methyl
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jasmonate, or mixtures thereof Conditionally male-stenle plants may be jasmonate-deficient (including, for example, plants having at least one mutation at a FAD locus, but are not necessarily so (e g , Arabidopsis line CS2338) Conditionally male-fertile plants may, for example, have one or more mutations 5 of a FAD gene and/or one or more mutations in the locus that causes male-stenlity in CS2338
Another aspect of the present invention encompasses methods of identifying a conditionally male-fertile plant by applying to a male-stenle plant an effective amount of a composition comprising jasmomc acid or a related 10 compound and detecting whether male fertility is thereby restored to the plant ^uch methods can be used to screen a population of male-sterile plants produced by mutagemzing fertile plants by conventional methods (e g , by chemical mutagenesis, irradiation, gene disruption by mobile genetic elements, antisense expression, cosuppression, gene replacement, etc )
IS Another aspect of the present invention encompasses methods is the use of conditionally male-fertile plants for the production of hybrid plants in plant breeding programs During each cycle of the repeated cycles of the inbreeding phase, a conditionally male-fcrtde plant is made fertile by application of a composition that includes an effective amount of jasmomc acid or a related 20 compound and the plant is self-fertilized to produce an inbred plant Then,
during the production phase, the inbred plant (which is allowed to exhibit the male-stenle phenotype) is crossed with a second plant to producc a hybrid plant
DETAILED DESCRIPTION OF THE INVENTION 25 As used herein, the term "conditionally male-fertile" plant refers to a plant having a dominant or recessive male-sterile trait (which is inherited in a Mendehan manner), whereby the fertility of the plant can be recovered by the administration of exogenous jasmonate or a related compound(s)
An "effective amount" of a composition comprising jasmonate or a 30 related compound(s) is an amount that, when applied to a conditionally male fertile plant as described herein, restores the fertility of the plant to a level acceptable for plant breeding purposes
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Identifying Conditionally Male-Fertile Plants
The present invention provides methods for identifying conditionally male-fertile plants varieties that are useful, for example, for hybnd breeding programs Such conditionally male-fertile plants may be found in any plant 5 species in which jasmonate or related compounds are required for pollen fertility including, but not limited to, rapeseed, canola and other Brassica species, soybean, wheat, barley, com, sunflower, tomato, tobacco, cotton, and nee
Lines of male-sterile plants have been reported for a large number of plant species See, e g , "Genie Male Sterility," In Kaul, Male Sterility in 10 Higher Plants, Spnnger-Verlag, Berlin, Heidelberg, pp 15-96, 1988
It is possible to determine whether such male-stenle lines are, in fact, conditionally male fertile by determining whether the lines recover male fertility upon administration of jasmonate or a related compound The Examples below demonstrate the effectiveness of such an approach As described below for line 15 CS2338, it is practical to grow progeny from a plant that is heterozygous for a particular male-stenle trait Once homozygous (male-stenle) segregants have been identified among the progeny by their inability to set seed (or by ether cnteria), then these sterile individuals are treated with jasmonate or a related compound If the plants produce seed after jasmonate treatment, but not after 20 control treatments lacking jasmonate or a related compound, then it can be concluded that these plants come from a line that contains a conditionally male-fertile trait
Many conditionally male-fertile lines according to the invention are substantially deficient in jasmomc acid in their vegetative tissues as the result of 25 a mutation affecting the biosynthesis of jasmomc acid from a-linolenate Such mutants can be identified by conventional techniques for identifying a deficiency in jasmomc acid or the buildup of a precursor compound, e g , by enzyme assays, radiotracer studies, gas chromatography (Creelman et al , Proc Natl Acad. Set USA 89 4938-4941, 1992), high performance liquid chromatography, 30 or other techniques In addition, because jasmomc acid and some related compounds are involved in the defense of plants from insect attack, it is possible
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to visually screen male-sterile plants for conditional male fertility on the basis of their greater susceptibility to insect attack and damage
A conditionally male-fertile fad3-2 fad7-2 fad8 triple mutant of Arabidopsis thaltana and of the Arabidopsis line CS2338 (discussed below) 5 displayed normal anther and pollen development up until the very last stages of pollen maturation and/or dehiscence of the anther locules Visual inspection and other experimental procedures (e g , as described in Regan and Moffatt, Plant Cell 2 877-889, 1990) can thus be used to screen male-sterile plants for lines that display anther and pollen development similar to these conditionally male-fertile 10 lines and are thus more likely to be conditionally male-fertile
Plant breeding programs incorporating male-sterile plants for the production of hybrids are described, for example, in U S Patent Nos 4,654,465, 4,727,219, 5,356,799, and 5,436,386
Production of Conditionally Male-Fertile Plants
Another embodiment of the present mvenuon involves the production of conditionally male-fertile plants by conventional mutagenesis techniques Such conditionally male-fertile plants are produced by mutations that eliminate or substantially reduce the expression of genes encoding, for example enzymes for 20 the biosynthesis of jasmonate from a-hnolenate, polypeptides that arc responsible for the regulation of such genes, receptors for precursors ot jasmonate, polypeptides necessary for intra- or intercellular transport of jasmonate or related compounds or the intermediates or products of its biosynthesis or metabolism Such genes include, but are not limited, to FAD (fatty acid gesaturation) genes 25 (Somerville and Browse, Trends in Cell Biol 6 148-153, 1996), including, but not limited to, an <i>-3 fatty acid desaturase, e g , FAD3 (Arondel et al, Science 258 1353-1355, 1992), FAD7 (Yadav et al , Plant Physiol 103 467-476, 1993), and FAD8 (Gibson et al , Plant Physiol 106 1615-1621, 1994), lipoxygenases, e g , LOX1 (Melan et al , Plant Physiol 101 441-450, 1993), and LOX2 (Bell 30 and Mullet, Plant Physiol 103 1133-1137, 1993), allene oxide synthase (Song et al , Proc Natl Acad Set USA 90 8519-8523, 1993), allene oxide cyclase, and
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the gene responsible for the conditionally male-fertile phenotype in the line CS2338 (discussed in the Examples below)
Mutagenesis techniques useful for producing conditionally male-sterile plants include, but are not limited to, genetic approaches such as the use of 5 ionizing radiation (e g , irradiation with X-rays or gamma rays) or chemical mutagens, gene disruption by mobile genetic elements such as transposons, or genetic engineering techniques, include targeted gene disruption or gene replacement (mediated by homologous recombmation or other means), antisense expression, or cosuppression of an appropriate gene, or other conventional 10 techniques
Targeted gene disruption will produce a recessive conditionally male-fertile trait similar to those identified from mutant screens By contrast,
antisense expression or cosuppression will produce a dominant trart, causing F1 hybrids to express the conditionally male-fertile phenotype This problem can be IS overcome in a breeding program by incorporating into the nuclear genome of the male line a second gene that is functionally equivalent to the suppressed gene but which has a DNA sequence that is sufficiently different from the suppressed gene to prevent its own suppression (e g , homologs of the inactivated gene from the same plant species or from other organisms or genes that have been mutagemzed 20 in vitro to introduce silent or conservative mutations that do not substantially interfere with biological function)
As described in greater detail in the Examples below, a fad3-2 fed?-2 fad8 triple mutant of Arabidopsis thaliana in which a-linolemc acid was substantially eliminated in all tissues was found to be male-stenle but was 25 otherwise apparently normal with regard to its growth rate and other characteristics, at least under laboratory conditions Suipnsingly, both a-linolemc acid and jasmomc acid, a product of a-linolemc acid metabolism, restored fertility to the triple-mutant plants The restoration of fertility to the triple-mutant plants was especially surprising m light of previous experiments in 30 which the application of jasmomc acid (10 fiM to 400 /zM) and the methyl ester of jasmomc acid (saturating concentrations) for ten days to the same fad3-2
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fad7-2 fad8 triple mutant failed to complement the male-stenle phenotype of the mutants (McConn, Ph D Thesis, Washington State University, December 1994)
In an effort to obtain more suitable conditionally male-stenle traits that might form the basis for hybnd breeding schemes, a survey of 24 presently 5 available male-stenle Arabidopsis lines resulted in the discovery of one line,
designated CS2338, that segregated male-stenle individuals that could be induced to set seed by the administration of jasmomc acid to developing flower buds This line was subsequently established to have wild-type levels of a-linolemc acid in leaf and flower tissues This result demonstrated the existence ot 10 conditional male fertility that is contingent on the admimstrauon of jasmomc acid or related compounds is not limited to lines that lack a-linolemc acid
Metabolic and developmental processes are highly conserved among higher plants, many of the genes controlling such processes are also highly conserved in both sequence and function Mutations in one or more genes IS encoding an enzyme in the biochemical pathway for jasmomc acid biosynthesis, including mutations analogous to those described herein for Arabidopsis thahana, produce conditional male fertility in other plant species Such genetic variants form the basis for successful hybnd breeding of these crops Mutations in genes encoding regulators of the pathway for the biosynthesis or intracellular or 20 intercellular transport of jasmonate or related compounds or the intermediates or products of its biosynthesis or metabolism also give rise to conditional male fertility
It is possible that in other plant species genotypes substantially deficient in a-linolenate would be unsuitable for hybnd breeding because of the 25 considerable breeding effort required to identify suitable alleles and to backcross them into agronomically suitable lines In Arabidopsis, for example, the substantial elimination of a-linolenate required the accumulation of at least three genetic loci A substantial breeding effort would be required to identify suitable alleles and to backcross them into agronomically suitable luies In addition, it is 30 possible that male sterility will require a deficiency in a-linolenate in all tissues Finally, genes encoding linoleoyl desaturases are codominant in Arabidopsis and
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other plant species studied (Ohlrogge et al , Biochim Biophys Acta 1082 1-26, 1991)
The biochemical steps in the synthesis of jasmonic acici from a-linolenate have been described (e g , Vick, In Moor, ed , Lipid Metabolism m 5 Plants, CRC Press, Boca Raton, FL, 1993) For additional fiiromiauon regarding plant hpid metabolism, see Plant Lipid Metabohsn, ed Kader and Mazliak, Lluwer Academic Publishers, Dordrecht, 1995, particularly Browse et al , pp 9-14 therein, which discusses a triple-mutant line of Arabidopsis thahana that is substantially deficient in of-linolemc acid See also -VlcConn, Ph D 10 Thesis, Washington State University, December 1994
Jasmonic id and Related Compounds may apply to the plant an effective amount of a composition comprising 15 jasmonate or a "related compound "
jasmomc acid in restoring the fertility of conditionally male-fertile plants, preferably compounds that are structurally related to jasmomc acid, including, but not limited to, compound having the formula wherein Rt is H or a lower alkyl chain of 1 to 6 carbon atoms, R} and R} are 25 independently selected from H, -OH, *0, or a lower alkyl having from 1 to 6 carbon atoms; and wherein the compound is optionally single or double bonded at one or more of Cj Cj, Cj C41 Cj, Cj C$, Cg Ct, Cy Cu, or Cu C|j, and where additional -OH groups may be optionally located at one or more of Ct, C„, or CI3 Representative compounds that are structurally related to jasmonate 30 include, but are not limited to cucurbic acid 7-zjo-jasmonic acid,
9,10-dihydrojasmoiuc acid, 2,3-didehydrojasmomc acid, 3,4-didehydrojasmomc acid, 3,7-dtdehydrojasmonic acid, 4,5-didehydrojasmomc acid,
In order to restore fertility to conditionally male-fertile plants, one
The term "related compound" includes compounds that function like
•x.
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,6-didehydrojasmomc acid, and derivatives thereof including lower alkyl esters and stereoisomers Preferred related compounds include jasmomc acid [(3R,7S)-jasmomc acid], the methyl ester of jasmomc acid ("methyl jasmonate"), and mixtures thereof, including racemic mixtures containing related enantiomers 5 and/or diastereoincrs
Also included among these "related compounds" are metabolic precursors of jasmomc acid and related compounds, including, but not limited to, a-linolemc acid (9Z, 12Z, 15Z octadecatnenoic acid),
13(S)-hydroperoxylinolenic acid (13-hydroperoxy-9Z,llE,15Z-octadecatnenoic 10 acid), allene oxide (12,13(S)-epoxy-9Z,ll,15Z-octadecatnenoic acid),
12-oxo-phytodienoic acid (8-[2-cw-2'-pentenyl)- 3-oxo-cyclopent-4-enyl]octanoic acid) (Blechert et al, Proc Natl Acad Sci USA 92 4099-4105, 1995), (lS,2S)3-oxo-2-(2'~pentenyl)cyclopentane-octanoic acid, (lS,2S)3-oxo-2-(2'-pentenyl)cyclopentanehexanoic acid, (lS,2S)3-oxo-2-(2'-15 pentenyl)cyclopentanetetranoic acid Such compounds may act directly as fertility-restoring agents or may be metabolized in the plant to fertility restoring agents Structurally-related compounds also include conjugates of these compounds with other moieties that do not interfere with fertility-restoring activity, including, for example, mono- or polysaccharides, amino acids, or 20 polypeptides
"Related compounds" also include other compounds that restore at least partial fertility to conditionally male-fertile plants, e g , compounds that interact with a jasmomc acid receptor, such as coronatine and coronofacic acid, or that affect other molecules involved in the jasmonate signaling pathway, 25 including proteinase inhibitors such as bestatin (Schaller et al , Plant Cell 7 1893-1898, 1995)
Administration of Compositions That Include Jasmonate or Related Compound^ Jasmonate acid and related compounds may be administered to a plant 30 by any conventional means, including direct applicauon to the flowers or other tissues of the plant, e g , by painting or spraying on the flowers or other plant
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tissues a composition that includes jasmonate or related compound(s) or a mixture thereof
For direct application, a composition that includes jasmonate or a related compound may be painted or sprayed onto the flower, flower bud, or 5 other plant tissue, for example The composition may be a solution of jasmonate or a related compound in water (or another suitable non-phytotoxic solvent such as glycerol) and may optionally include a wetting or sticking agent, e g , a low concentration of a suitable surfactant The composition may be applied directly to a flower or other plant tissue by any conventional means, such as by painting, 10 misting, spraying, drenching, etc
Jasmomc acid or a related compound can also be "applied" to plant tissue via airborne transmission in which volatile source of the inducing agent is placed in the vicinity of the plant tissue and the agent is allowed to diffuse or disperse through the atmosphere to contact the plant tissue. The volatile source 15 may be, for example, plant materials which naturally produce the inducing agent or a volatile solution of the agent, such as a solution comprising a volatile solvent and the inducing agent Suitable solvents for this purpose include organic solvents, e g , alcohols (e g , methanol and ethanol), or water The manner in which the volatile source of the inducing agent is placed in the vicinity of the 20 plant tissue to be treated is not critical to the practice of the invention For example, the inducing agent may be placed in an open container in the vicinity of the plant tissue, on a matrix support, such as a fiber matrix, in the vicinity of the plant tissue
Ajnounts of jasmomc acid or related compounds effective to induce 25 fertility m treated plants of a given male-sterile luie depend on many factors, including the nature, environment and condition of the plants to be treated, the method of contact of the inducing agent with plant tissue to be treated and other factors Preferably, a solution of jasmomc acid or a related compound for direct application comprises from about 1 pg/ml to about 100 mg/ml of the inducing 30 agent, more preferably from about 1 ng/ml to about 10 mg/ml of the inducing agent, and most preferably from about 1 fig/ml to about 1 mg/ml of the inducing agent Experience with the mutants described herein suggests that a higher
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jasmonate concentration will be required to induce fertility if the jasmonate is sprayed onto a plant tissue rather than painted on the tissue
Additional information regarding methods for applying jasmomc acid and related compounds to plants may be found in WO 91/18512
Jasmomc acid and related compounds induce compounds that act in the defense of plants from insect attack It is possible that conditionally male-ferfile plants that are rendered fertile by the administration of exogenous jasmomc acid or related compounds will be more susceptible to insect damage (Farmer and Ryan, Proc Natl Acad Sci USA 87 7713-7716, 1990) Protection against insect damage can be provided through the use of msecticidal treatments Application of jasmonic acid or related compounds may itself reduce insect damage, as described m WO 91/18512, which is incorporated herein by reference
The foregoing may be better understood in connection with the following Examples
EXAMPLE 1 Sterility and Flower Morphology of Lmolenate-Deficient Arabidopsis
The biophysical reactions of light harvesting and electron transport during photosynthesis take place in a uniquely constructed bilayer membrane, the thylakoid In all photosynthetic eukaryotcs, the complement of atypical glycerolipid molecules that form the foundation of this membrane is characterized by sugar headgroups and a very high level of unsaturation in the fatty acid chains that compose the central portion of the thylakoid lamella bilayer For example, monogalactosyldiacyl-glycerol, the major thylakoid lipid, typically contains more than 90% of a-linolemc acid (18 3) or a combination of 18 3 and hexadecatrienoic (16 3) acids, depending on the plant species (Jamieson and Reid, Phytochertustry 10 1837-1843, 1971) These very high levels of trienoic fatty acids are noteworthy because free radicals that are by-products of the photosynthetic light reactions stimulate oxidation of polyunsaturated fatty acids Since this might be expected to mediate against a high degree of unsaturation, it has been inferred that there is a strong selective advantage to having such high levels of trienoic acids in the thylakoid These lipid structures, it is reasoned, must have some critical role in maintaining photosynthetic function
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There are two distinct pathways in plant cells for the biosynthesis of glycerolipids and the associated production of polyunsaturated fatty acids (Browse and Somerville, Ann Rev Plant Physiol Plant Mol Biol 42 467-506, 1991) Both pathways are initiated by the synthesis of 16 0-ACP and 18 1-ACP by the 5 combined action of a Type II fatty acid synthase and a soluble stearoyl-ACP desaturase located m the chloroplasts or other plastids The "prokaryotic pathway" located in the chloroplast inner envelope uses 18 1-ACP and 16 0-ACP for the sequential acylation of glycerol-3-phosphate and synthesis of glycerohpid components for the chloroplast membranes The "eukaryotic pathway" involves 10 export of 16 0 and 18 1 fatty acids from the chloroplast to the endoplasmic reticulum and their incorporation into phosphatidylcholine and other phospholipids that are the principal structural lipids of all the membranes of the cell except for the chloroplast In addition, the diacylglycerol moiety of phosphatidylcholine can be returned to the chloroplast envelope and used as a IS second source of precursors for the synthesis of chloroplast glycerolipids
In each pathway, further desaturauon of 16 0 and 18 1 occurs only after these fatty acids have been incorporated into the major membrane lipids Thus, most of the plant desaturases responsible for the synthesis of 18 3 and 16 3 are integral membrane proteins that utilize glycerolipids as substrates In 20 Arabidopsis, for example, there are three desaturase enzymes that mediate the conversion of 18 2 and 16 2 acyl groups to 18 3 and 16 3 The FAD7 and FADS genes encode two chloroplast isozymes, both of which can recognize as a substrate, either 18 2 or 16 2 attached to any of the chloroplast glycerolipids The FAD3 gene product, localized predominantly in the endoplasmic reticulum, 25 utilizes 18 2 on phosphatidylcholine as its major substrate although it is possible that it also acts on 18 2 groups of other phospholipids (Browse et al , J Biol Chem 268 16345-16351, 1993)
The operation of parallel pathways of glycerolipid desaturation complicates the task of eliminating trienoic fatty acids from the plant For 30 example, fad7-2 fad8 double mutant plants contain no 16 3 but approximately 17% 18 3 in their chloroplast membranes (McConn et al , Plant Physiol 106 1609-1614, 1994) The extrachloroplast membranes in leaves of fad3 mutant
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plants contain considerable 18 3 because 18 2 lipid can be transferred to the chloroplast on the eukaryotic pathway, desaturated by the FAD7 and FAD8 enzyi.ies, and then returned to the endoplasmic reticulum and other extrachloroplast membranes (Browse et al , J Biol Chem 268 16345-16351, 5 1993)
To investigate the relevance of 18 3 and 16 3 fatty acids to the biology of higher plants, crosses were made between lines of Arabidopsis thahana (L ) Heynh fad7-2 (McConn et al, Plant Physiol 106 1609-1614, 1994) and fad8 (ibid) and between fad3-2 (Browse et al , J Biol Chem 10 268 16345-16351, 1993) and fad7-2 (Additional mutant lines were created using fad7-l, Browse et al , Plant Physiol 81 859-864, 1986) The ecotype of Arabidopsis used in generating the triple-mutant plants and as the wild type controls was Columbia Wild-type and mutant Arabidopsis plants were grown on commercial potting mix in controlled environment chambers at 22 °C and 15 continuous fluorescent illumination of 140 /zmole quanta/m2/s Earlier results had indicated that thcfad3-l and fad7-l mutations probably represent leaky alleles, each of which retains a small amount of the relevant desaturase activity (Browse et al , J Biol Chem 268 16345-16351, 1993, McConn et al , Plant Physiol 106 1609-1614, 1994)
The F2 progeny from these crosses were screened by gas chromatographic analysis and fad.7-2 fad8 and fad3-2 fad7-2 double mutant plants were identified To screen the leaf fatty acid compositions of individual F2 progeny, samples of leaf tissue were rapidly immersed m liquid nitrogen, ground to a fine powder, extracted and analyzed for their content of trienoic fatty acids 25 using gas chromatography essentially as described by Miquel and Browse (J Biol Chem 267 1502-1509, 1992)
The two double mutant plants were crossed, and an F, plant derived from a cross between the two double mutant plants was allowed to self-pollinate The resulting seeds were germinated The leaf fatty acid compositions of 30 individual F2 progeny were examined for their content of trienoic fatty acids Of 240 F2 plants analyzed, 17 contained no detectable 16 3 or 18 3 (detection limit approximately 0 1 % of total) while the remaining plants exhibited a range in the
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proportion of total trienoic fatty acids from 10% to 40% (Table 1) Such a segregation pattern is a good fit to the Mendelian expectation (chi-squared = 0 284, p > 0 5), indicating that the homozygous fad3-2 fad7-2 fad8 progeny were not selected against during embryogenesis or seed germination 5 Visual comparison of wild-type Arabidopsis and fad3 fad.7-2 fad8
tnple-mutant plants grown for four weeks at 22°C under continuous illumination (140 pmol quanta/m^s1) revealed no striking difference in vegetative growth and development between the triple mutant and wild type
In later experiments, tnple-mutant plants were identified by fatty acid 10 analysis from among the progeny of plants that contained just one wild-type allele at the Jad8 locus, that is, fad3-2(-l-) fad7-2(-/~) fad8(+/-)
Under the growth conditions used, wild-type plants began to bolt about three weeks after sowing of the seed and flowers are produced continuously for 4-6 weeks thereafter until the plant became senescent The 15 growth rate of linolennte-deficient tnple-mutant plants (genotype fad3-2 fad7-2 fadS) was indistinguishable from the wild-type plant during vegetative development at this temperature (FIG 1) and tnple-mutant and wild-type plants displayed a similar tuning for bolting and the start of flowering
Moreover, there was no difference between wild-type and mutant 20 plants with regard to increases in shoot fresh weight or the overall capacity of photosynthetic processes at 25 °C Photosynthesis appeared to be reduced relative to wild-type at low temperatures, but tnple-mutant plants grew well at temperatures as low as 6°C
Because tnenoic fatty acids are invariably abundant components in 25 membranes of photosynthetic eukaiyotes, it was surpnsing that Arabidopsis mutants lacking 16 3 and 18 3 would be viable, and more surprising yet that the growth rate of these mutants would be the same as that of wild-type plants These findings indicate that the highly unsaturated tnenoic fatty acids are not absolutely required as components of the thylakoid or other cell membranes 30 The only essential requirement for 18 3 in the plant life cycle appears to be as a substrate for the pt !—<ion of octadecanoid compounds, including jasmonic acid
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TABLE 1
The Linolenic Acid Content of Leaves and Floral Organs of Wild-Type and Mutant Arabidopsis Plants
Plant Genotype
Floral Organs Leaves Sepals Petals Carpels
Anthers
| Wild Type
46.4*
43.5
32.8
36.7
39.7
fad3-2 Jfad7-1 fadB
.7
4.9
2.2
3.0
2.2
fad3-2 fad7-l/fad7-2 fade
3.1
2.8
1.1
1.7
1.0
i fad3-2 fad!-2 fad8
<0.1
<0.1
<0.1
<0.1
<0.1
1 Data are mole % of total fatty acids (n = 6)
PCT/US96/1S131
EXAMPLE 2 The Arabidopsis fadS-2 fadl-l fadS Triple Mutant is Male Sterile An unanticipated consequence of the lack of 18 3 and 16 3 lipids was the fact that the tnple-mutant plants were profoundly male sterile under all the growth conditions used 5 The first generation of homozygous fad3-2 fad7-2 fad8 plants dd not set any seed, and flowers of the mutant retained their petals longer than wild-type flowers The failure of the petals to senesce is typical of such sterile flowers To determine the nature of flower sterility, reciprocal crosses were performed between wild-type and tnple-mutant plants Using anthers from wild-type 10 flowers, it was always possible to pollinate the mutant and produce mature seeds In contrast, anthers from mutant flowers were unable to induce seed set on emasculated wild-type flowers Closer examination of wild-type and mutant flowers revealed that the locules of the mutant anthers had not dehisced to deposit pollen on the stigmatic surface Manual disruption of the anther locules 15 to release the enclosed pollen did not result in any seed set
Eventually, the majority of mutant anthers did open, but separation of the stomium occurred later and the outward bending of the locule walls, which leads to pollen release in the wild-type, did not occur Scanning electron micrographs revealed that pollen in the mutant anther appeared morphologically 20 normal For scanning electron microscopy, mature flowers were harvested and fixed overnight in 3% glutaraldehyde at 4°C Samples were then washed three tunes for 10 minutes each on 0 1 M PIPES pH 7 2 and incubated m 2% (v/v) 0s04 for two hours, then dehydrated through a graded ethanol series (50, 60, 70, 80, 90, 95, 100%, v/v) Once in 100% ethanol, the samples were transferred to 25 a Samdn-PVT-3D drying apparatus and critical point dried in C02 (Boyde, Scanning Electron Microscopy 2 5945-5951, 1978, Cohen, Scanning Electron Microscopy 2 303-323, 1979) The specimens were then affixed to stubs with paraffin, coated with gold in argon with a Technics Hummer V sputtering device (Echlin, Scanning Electron Microscopy 1 79-80, 1981) and viewed on a Hitachi 30 S-570 scanning electron microscope Two independent preparations were examined
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The observation that tnple-mutant progeny were produced m a Mendehan ratio from either fad3-2(-¥l-) fad7-2(+l-) fad8 (+/-) or fad3-2{-l-) fad7-2(-!-) Jad8 (+/-) (where " + " indicates a wild-type allele and indicates a mutant allele at each locus in the diploid genome) parents indicates that the 5 genotype of the maternal tissue (rather than the genotype of the segregating,
haploid microspores) mediates the male-stenle phenotype and that very low levels of 18 3 are probably sufficient to ensure fertility
In subsequent experiments, flowering triple-mutant plants were grown under different environmental conditions without restoring fertihty to them 10 These conditions included light intensity ranging from 100-150 and 300 ft mole quanta/mzs, photopenods including continuous light, 12 hr light, 12 hr darkness, and 10 hr light, 14 hr darkness, and a variety of temperatures from 5°C to 22°C The male sterility of the fad3-2 fad7-2 fad8 tnple-mutant plants persisted under a range of environmental conditions 15 From a total of 15,000 flowers on more than 150 untreated tnple-
mutant plants, not a single seed of the parental genotype fad3-2 fad.7-2 fad8 was ever recovered This represents a seed-production capability that was less than 10s of wild-type plants, based on typical average values for seeds produced per flower for wild-type Arabidopsis under the growth conditions used 20 Successful pollination and fertilization of flowers on fad3-2 fad.7-2
fad8 plants were readily achieved using pollen from wild-type plants This observation indicates that it was the inale-fertility of mutant plants which was specifically affected
EXAMPLE 3. Germination and Viability of Pollen from the Tnole Mutant Pollen viability was assessed by double staining pollen grains with fluorescein diacetate and propidium iodide essentially as desenbed by Heslop-Hamson and Heslop-Harnson (Stain Technol 45 115-120, 1970) and by Regan and Moffatt (Plant Cell 2 877-889, 1990) Equal amounts of fluorescein 30 diacetate and propidium iodide solutions were added to freshly isolated pollen The pollen was transferred to a glass slide, covered with a coverslip, and viewed
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under ultraviolet light using filter block 13 with excitation filters BP450-490, dichromatic minor RKPS10 and suppression filter LP520
Pollen from wild-type Arabidopsis plants was fertile both in self-pollination, which usually occurs as a flower opens, or in pollination of flowers 5 of another plant such as the male-stenle tnple mutant when pollination was performed manually Pollen released by disruption of non-dehiscent locules from triple-mutant flowers failed to induce seed set after transfer to the stigmas of emasculated wild-type flowers
To determine the viability of pollen produced on the wild-type and 10 fad3-2 fad7-2 fad8 mutant plants, the contents of wild-type and mutant anthers were released into osmoticum on microscope slides and double stained with fluorescein diacetate and propidium iodide following the method of Regan and Moffatt (Plant Cell 2.877-889, 1990) with some alterations A stock solution of 2 mg/ml fluorescein diacetate was made in acetone and added drop wise to 17% 15 sucrose (w/v) Equal amounts of fluorescein diacetate and propidium iodide solutions were added to freshly isolated pollen The pollen was transferred to a glass slide, covered with a coverslip, and viewed under ultraviolet hght using filter block 13 (Leitz) with excitation filters BP450-490, dichromatic mirror RXP510 and suppression filter LP520 (all from Leitz) 20 Fluorescein diacetate, a vital stain, is taken up by living cells and converted to impermeant fluorescein, which emits a green fluorescence under ultraviolet light (Heslop-Hamson and Heslop-Hamson, Stain Technology 45 115-120, 1970) Propidium iodide is excluded from living cells but labels dead cells with a red-orange fluorescence under ultraviolet light (Regan and 25 Moffatt, Plant Cell 2 877-889, 1990)
The viability of pollen from mutant plants was very low relative to wild-type pollen Counting of live and dead pollen (based on their fluorescence) in 23 microscope fields in four independent experiments indicated an average of 11% viable pollen from the fad3-2 fad7-2 fad8 mutant compared with 84% from 30 the wild-type
A significant proportion of the pollen from the tnple-mutant plants was alive at maturity, but this pollen did not induce seed set when removed from
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anthers and applied to the stigmas of mutant or emasculated wild-type flowers Apparently, the viability of pollen grains from tnple-mutant plants was considerably reduced compared with pollen grains from wild-type plants However, pollen was produced in amounts greatly in excess of the minimum 5 needed to fertilize all the ovules in a typical flower, m principle, the 11% viable pollen released from the disrupted locules of tnple-mutant plants should have effected at least some fertilization and seed production when applied to the stigmas of mutant or emasculated wild-type flowers
To further characterize pollen produced on the wild-type and tnple-10 mutant plants, the ability of wild-type and mutant pollen to germinate and produce a pollen tube was measured in vitro Pollen from wild-type Arabidopsis plants normally germinates with high efficiency on a germination medium in vitro and most grains produce a long pollen tube When germination occurs on the stigma of a flower, this pollen tube grows down within the conducting tissue IS of the style to facilitate transfer of two sperm cells for the double fertilization of the female gametophyte (Mascarenhas, Plant Cell 5.1303-1314, 1993)
Wild-type and tnple-mutant plants were grown side-by-side m four separate experiments Pollen was isolated from mature flowers by gently releasing them from the anther locules into 17% (w/v) sucrose The liberated 20 pollen was then placed onto plates of pollen germination medium, consisting of 17% (w/v) sucrose, 2 mM CaCl2, 1 65 mM H3B03 at pH 7 (Preuss et al , Genes and Development 7 974-985, 1993) and solidified with 6% (w/v) agar Pollen was incubated for 12 hours at room temperature then analyzed for pollen tube formation
A total of more than 1,000 pollen grains from wild-type plants viewed in twenty microscope fields showed an average germination of 82%, a figure agreeing closely with the determination of average viability In contrast, only eight germinated pollen grains were observed in twenty-eight microscope fields (approximately 1,400 grains) of pollen from mutant plants (germination less than 30 0 6%) It was consistently observed that pollen grains from mutant plants that did germinate produced pollen tubes that were less than one-third the length of pollen tubes produced by wild-type pollen
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EXAMPLE 4. Development of Mutant and Wild-Type Pollen
To examine possible differences in the development of wild-type and mutant pollen, young floral buds were fixed and cleared by a technique that permitted examination and optical sectioning of intact tissues, as described in 5 Herr, Amer J. Bot 58 785-790, 1971) Flowers were fixed m FPA50 (Sass, Botanical Microtechnique, Ames, Iowa, Iowa University Press, 1958) overnight at room temperature and dehydrated through a graded ethanol senes (50, 60, 70, 80, 90, 95, 100%, v/v), then cleared in Herr fluid (Herr, Amer J Bot 58 785-790, 1971) at room temperature After 48 hours in Herr fluid, the 10 flowers were dissected, immersed in fluid, and viewed on Raj slides Both phase-contrast and differential interference-contrast optics were used to view clearings on a Lsitz (Wetzlar, Germany) Austoplan microscope Images were recorded on Kodak Technical Pan film and Ektachrome color slide film
Pollen development in the mutant followed a course that was very 15 similar to that of wild-type pollen Pollen mother cells undergoing the first division of meiosis formed haploid progeny which, following the second meiotic division, become organized into tetrads of microspores encased in a callose wall The individual microspores were released by the action of a callase enzyme secreted by cells of the parental tissues The layer of specialized tapetai cells 20 that provides many of the nutrients and other factors (including the extracellular callase) required for pollen development persisted until late in development Shortly before flower opening and the associated dehiscence of the wild-type anthers, the tapetum broke down in a process that resulted in deposition of a lipid-based material, sporopollemn, on the exine of the mature pollen grains 25 The fluorochrome stain 4,,6-diamidino-2- phenylindole (DAPI), which binds specifically to double-stranded DNA, was used to stain pollen from wild-type and mutant plants that bad been removed from anthers at stages immediately before or immediately after flower opening DAPI staining was performed following the procedure of Coleman and Goff (Stain Technology 60 145-154, 30 1985) The pollen was double stained with fluorescein diacetate and propidium iodide to show viable (blue-green) and dead (red-orange) pollen grams DAPI-stained pollen was viewed with ultraviolet light using filter block A, with
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excitation filters BP340-380 dichromatic mirror RKP400 and suppression filter LP430 (all from Leitz) (Staining for DNA with mithramycin m the same manner provided similar results )
The majority of pollen from both wild-type and mutant plants 5 exhibited three fluorescent spots corresponding to one vegetative nucleus and two smaller generative nuclei (Stanley and Lmskens, Pollen Biology, Biochemistry and Management, New York, Spnnger-Verlag, 1974), indicating that the pollen had matured to the tnnuclear stage (Stanley and Linskens, Pollen Biology, Biochemistry and Management, New York, Spnnger-Verlag, 1974) 10 Because pollen grains of Arabidopsis become tnnucleate only during the final stages of development, these results strongly suggested that male-sterility m the fad3-2 fad.7-2 fad8 tnple-mutant plants was determined by events which occur very late m pollen development It appeared that all essential processes dunng the earlier stages occurcd in the mutant plants as in the wild-15 type
EXAMPLE 5 Restoration of Fertility of the Tnole Mutant with Exogenous q-Linolenic Acid
The observation that fad3-2 fad7-l fad8 plants were fully fertile
despite having less than 5% 18 3 m their tissues suggested that the threshold requirement for 18 3 must be very low We therefore treated wild-type and tnple-mutant plants dunng flowering by spraying them with a 0 1% (w/v)
solution of the normal plant isomer of 18 3 (A9,12,15 all cis) as the sodium salt
Other plants were treated with sodium salts of either 18 2 or a second isomer of
hnolenate, 7I8 3 (A6,9,12 all cis), at <x>ntrols
Sodium soaps of a-licolemc acid (9Z, 12Z, 15Z-octadecatnenoic acid) and other fatty acids (Nuchek, Elysian, MN) were dissolved in water to make 0 1 % (w/v) solutions The solutions were applied either by spraying plants that were flowering or by painting the solutions onto unopened flower buds
Three pots containing wild-type Arabidopsis plants and three pots containing fad3-2 fad7-2 fad8 triple-mutant plants were grown for approximately four weeks under continuous light (140 jimole quanta/m2/s at 22°C) At this stage, enlarged, fertile siliques were present on all the wild-type plants The
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rnale-stenhty of the mutant was evident by the lack of any such enlarged siliques One pot of wild-type and one pot of mutant plants were sprayed with 2 ml of 0 1% solution of a-linolemc acid sodium soap each day for 10 consecutive days, during which nme the plants were kept on a regime of 12 hours light/12 hours 5 dark (at 22°C, 140 jumole quanta/m2/s dunng the lighted portion of the cycle) and sprayed at the start of the darkened portion of the cycle Other pots of wild-type and mutant plants were sprayed with the sodium soap of either linoleic acid (9Z, 12Z octadecadienoac acid) or -y-linolemc acid (6Z, 9Z, 12Z octadecatrtenoic acid) under the same conditions The plants were monitored for 10 silique production Mature siliques were harvested and the seed m them allowed to desiccate for one week before planting Leaves of the progeny were analyzed for their fatty acid composition by gas chromatography as desenbed above
Analysis of rosette leaves from tnple-mutant plants harvested at the end of the spraying treatment indicated that each of the exogenous fatty acids was IS taken up and incorporated into membrane glycerolipids to a level accounting for 5% of the total acyl groups Plants treated with linoleic or -y-linolenic acids did not produce any seeds However, plants sprayed with a-linoleruc acid yielded, on average, more than 300 seeds per plant When these seeds were germinated, all the progeny plants exhibited the fad3-2 fad7-2 fad8 fatty acid phenotype, 20 indicating that the seeds could not have resulted from fertilization by pollen from other plants In later trials, it was determined that painting the sodium salt of a-linolenic acid onto unopened flower buds of the fad3-2 fad7-2 fad8 triple mutant also resulted in seed set
Relatively low levels of exogenous 18 3 met the requirements for 25 pollen maturation and release However, pollen on untreated mutant plants died shortly before the flower buds opened To determine whether a-linolenate penetrated the unopened flower buds and became incoiporated into the tissues of the anthers, newly opened flowers that had been painted with a 0 1 % solution of the sodium salt of a-linolemc acid were dissected The a-linolenate content (as a 30 proportion of the total fatty acids) m leaves, roots, and flower tissues (sepals, petals, carpels, and anthers) was determined by gas chromatography as desenbed above after derivatization with 2 5% (v/v) H2S04 in methanol (Miquel and
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Browse, J Biol Chem 267 1502-1509,1992) The results for flowers of fad3-2 fad7-2 fad8 tnple-mutant plants treated m this way and for untreated mutant and wild-type flowers are shown m Table 2 As expected, wild-type plants produced seeds while untreated mutant plants were stenle On mutant plants that were treated with the sodium salt of a-lmolenate, flower buds that were not harvested for analysis did produce seeds Analysis of the different floral organs from similarly treated buds revealed that the a-lmolemc acid content of the anthers remained below the level of detection (<0 1%) In contrast, the sepals, petals, and carpels of these flowers, which are at least partly exposed at the bud stage, were found to contain measurable levels of a-linolenate
Mutant genotypes in which the anthers contain only 1-2% a-linolemc acid (compared with 44% for wsld-type plants) are fertile However, plants with a fad3-2 fad7-l/fad7-2 fadS genotype exhibited reduced seed set (approximately 25% fewer siliques than on wild-type and fad3-2 fad7-l fad8 plants), suggesting that a level of a-linolemc acid of 1 % in the floral organs may represent an approximate lower limit for fertihty As expected, infertile fad3-2 fad7-2fad8 flowers did not contain detectable a-linolemc acid Painting flower buds with the sodium salt of a-lmolemc acid restored fertility, but, even though the outer sepals contained 14% a-lmolemc acid, this fatty acid was not detected m the anthers of either unopened or opened flowers
These findings indicated that 18 3 is not specifically required in the anther tissue The cntical requirement for 18 3 is not likely to be as a structural component either of cell membranes or of the outer sporopollemn and tryphine layers of mature pollen Instead, these findings suggest that 18 3 in other flower organs is converted to a compound that regulates cellular functions in the anthers and thereby mediates maturation and release of viable pollen
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TABLE 2. The Linolenic Acid Content of Floral Organs, and Seed Set Observed m Wild Type and Mutant Arabidopsis Plants
Plant
Floral
Organs
Seed
Genotype
Sepals
Petals
Carpels
Anthers
Set
Wild Type
43.5 ± 1.9*
32.8 ± 1.1
36.7 ± 1.1
39.7 ± 0.9
+ 1
fad3-2 fad7-l fade
4.9 ± 0.3
2.2 ± 0.3
3.0 ± 0.4
2.2 ± 0.3
+ |
jfad3-2 fadl-l/fadl-2 fadB
2.8 ± 0.1
1.1 ± 0.3
1.7 + 0.1
1.0 ± 0.1
+/- I
fad3-2 fadl-2 fad8
<0.1
<0.1
<0.1
<0.1
1
.fad3-2 fadl-2 fadB
14.3 ± 0.9
1.7 ± 0.4
1.5 ± 0.7
<0.1
+ I
after application of 18.3
I
• Data are mole % of total fatty acids ± std error (n = 6). Unopaned flower buds on some fad3-2 fad7-2 fadB plants were painted with Nal8:3 at the start of the dark period for ten consecutive days At the end of this tine, the earliest-treated buds had already formed fertile siliques. Open flowers above these siliques were sampled for analysis.
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-n-
EXAMPLE 6. Restoration of Fertihty with Exogenous Jasmomc Acid
The octadecanoid signalling compounds, jasmomc acid and methyl jasmonate (available from Bedoukian Research Inc , Danbury, CT), activate wound responses in plants (Farmer and Ryan, Proc Natl Acad Set USA 5 87 7713-7716, 1990) and have been postulated to perform roles in several other developmental and environmental response processes (Sembdner and Parthier, Ann Rev Plant Physiol Plant Mol Biol 44 569-589, 1993) The structure and biosynthesis of jasmomc acid have intrigued plant biologists because of parallels to eicosanoid second messengers that are central to inflammatory responses and 10 other physiological processes m mammals (Creelman et al, Proc Natl Acad Set USA 89 4938-4941, 1992)
Jasmomc acid m plants is synthesized from a-linolemc acid (9Z, 12Z, 15Z octadecatnenoic acid), a component of plant cell membranes (which is presumably released from membrane lipids by the action of a phosphohpase A2) 15 by a pathway that is initiated by lipoxygenase Cyclization and /3-oxidation of the lipoxygenase product, 13(S)-hydroperoxylinolenic acid, result in the formation of jasmomc acid, which has a structure analogous in some respects to the prostaglandin E series of eicosanoids (Vick and Zimmerman, Plant Physiol 75 458-461, 1984) 13(S)-Hydroperoxylinolenic acid may also give nse to other 20 compounds, including products derived from a hydropeioxide lyase reaction sequence 3Z-hexen-l-ol, 2E-hexen-l-al, 3E-hexen-l-ol, and traumatic acid (Croft et al , Plant Physiol 101 13-24, 1993)
To determine whether jasmomc acid or other products of 18 3 metabolism could restore fertility to flowers of fad3-2fad7-2 fad8 triple-mutant 25 plants, plants were grown under tha conditions described above for approximately four weeks so that the sterility of the plants was evident by the absence of enlarged, developing siliques Aqueous solutions containing 2 jtraol/ml of jasmomc acid (7-epi-jasmomc acid. Cayman Chemicals, Ann Arbor, MI) were sprayed on unopened flower buds of these plants once a day for 10 30 days These solutions alternatively contained 0 1 % sodium linoleate as a surfactant or were made up in pure water without surfactant The results were comparable in either case
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fiy the end of the treatment period, the earliest-treated buds had already formed fertile siliques Mature seeds from these siliques were subsequently germinated and gave rise to plants with the fad3-2 fad.7-2 fad8 fatty acid phenotype (<01% a-linolemc acid) that were male sterile This result 5 indicates that the seeds produced were not the result of cross-pol 1 mat ion by another plant
Apart from the biochemical reactions that lead to the synthesis of jasmomc acid, there are other pathways for the further metabolism of a-lmolemc acid in plant tissues A partial list of the chemical compounds produced by these 10 pathways mcludes 3Z-hexen-l-ol, 2E-hexen-l-al, 3E-hexen l-ol, and traumatic acid (Croft et al , Plant Physiol 101 13-20, 1993) Each of these compounds was prepared as a 0 1 % aqueous soluuon using the sodium salt of linoleic acid (0 1%) as a surfactant and applied to fad3-2fad7-2 fadB triple-mutant plants as described above None of these compounds promoted any seed set on the mutant 15 plants These results suggest that restoration of fertility in the fad3-2 fad7-2 fad8 triple mutant is likely to be specific for intermediates in jasmomc acid synthesis and for compounds that are structurally-related to jasmomc acid
Flower buds on fad3-2fad7-2 fad8 triple-mutant plants that were treated with jasmomc acid opened mto flowers in which the anther locules 20 dehisced and released pollen in a manner similar to wild-type controls The fluorescein diacetate/propidium iodide double-staining technique was used to examine pollen from jasmonate-treated mutant flowers as well as from untreated mutant flowers and flowers from control wild-type plants The results (Table 3) indicate that jasmonic-acid treatment increased the viability of pollen produced 25 from 15% of the wild-type value to 80% of the wild-type value Although pollen germination tests were not conducted in this case, it is reasonable to assume that the restoration of male fertility, which was evidenced by the production of viable seeds, was mediated through the correction of defects in the maturation of pollen on the mutant plants by the applied jasmomc acid
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Table 3 Percentage viability of pollen from wild-type and fad3-2 fadl-2 fad& plants treated with fatty acids, jasmomc acid, or methyl 5 jasmonate
Pollen Source Viability
Wild-type 78 8%
fad3-2 fadl-2 fad% 12 2%
fad3-2 fadl-2 fad& + 18 3-7 13 8%
fadi-2 fadl-2 fadi + 18 3-a 61 8%
fadi-2 fadl-2 fadi + jasmomc acid 64 1%
fadi-2 fadl-2 fadS + methyl jasmonate 63 9%
Dunng some experiments mvolvmg regular daily applications of jasmomc acid solutions, it was occasionally necessary to omit applications for one or two days dunng the treatment When this was done, infertile siliques (undeveloped siliques containing no seeds) were sometimes observed in the 20 middle of the senes of fertile siliques that were produced following jasmomc acid treatment These findings indicated that jasmomc acid must be present in flower tissues during a relatively narrow window dunng pollen development Application of jasmomc acid outside this window (at the levels used in these experiments) was insufficient to cause the development and release of fertile 25 pollen
The only essential requirement for 18 3 in the plant life cycle appears to be as a substrate for the production of octadecanoid compounds, including jasmomc acid A role for jasmonate signaling in flower development has been inferred from characterization of coil mutants of Arabidopsis thaliana which are 30 also male-stenle The coil plants are resistant to the bacterial phytotoxin coronatme (whose structure is analogous in certain respects to that of jasmoiuc acid) and they fail to exhibit several typical responses when exposed to jasmonate (Feys et al , Plant Cell 6 751-759, 1994). It has been suggested that coil plants lack the jasmonate receptor, which acts also as the target for coronatine action 35 Some limited evidence has also existed for a role of jasmonate in pollen germination with low concentrations (107 to 10"6 M) stimulating germination and
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a higher concentration (105 M) inhibiting germination (Sembdncr and Parthier, Annu Rev Plant Physiol Plant Mol Biol 44 569-589, 1993)
The characterization of the fad3-2 fad7-2 fad8 mutant and the ability to chemically complement the mutant's male-sterile phenotype with exogenous 5 jasmonate establish an essential role for jasmonate in pollen maturation and anther dehiscence Although a large number of male-stenle mutant plants of various species have been isolated and characterized at the genetic level, the fad3-2fad7-2 fad8 triple mutant is one of the few for which the genetic defect has been defined at the biochemical level This mutant and others desenbed 10 herein and the associated chemical complementation assay provide powerful tools to further dissect the signalling processes involved in pollen and anther development
The lack of 18 3 and jasmomc acid appears to affect pollen development at a later stage than any previously-descnbed mutation that results IS in the production of aborted or dead pollen in Arabidopsis (Chaudhury, Plant Cell 5 1277-1283, 1993), Regan and Moffatt, Plant Cell 2 877-889 1990) The best estimate of the timing of jasmonate actions suggests that jasmomc acid or sodium-18 3 must be applied 12-24 hr before flower opening to ensure seed set, which corresponds to the middle of stage 12 as defined by Smyth et al (Plant 20 Cell 2 755-767, 1990) This timing would be consistent with a malfunction or cessation of pollen cell function in the 24 hr immediately prior to flower opening
The male-stenle phenotype m the triple mutant is controlled by the genotype of the sporophytic tissue This is true of the majority of male-stenle 25 mutants and the defects are often localized by direct or circumstantial evidence to processes occurring in the tapeturn, the anther cell layer that bounds the locule and is intimately involved in all aspects of pollen microspore development (Chapman, Int'l Rev Cytology 107 111-125, 1987, Goldberg et al , Plant Cell 5 1217-1229, 1993) It would seem reasonable to implicate the tapeturn as the 30 physiological source of jasmomc acid, thereby regulating final maturation processes in pollen grains However, 18 3 applied to the flower buds can bring about seed set without significantly raising the 18 3 content of the anther tissues,
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which strongly suggests that other floral tissues can be effective sources of eicosanoid compounds to complement the male-stenle phenotype m the mutant
Indeed, it is possible that sepals and other floral organs are sources of jasmomc acid in wild-type Arabidopsis and that the tapeturn is the target
In either case, transfer of jasmonate may occur by simple diffusion
This could be assisted by the fact that methyl jasmonate (which is normally present in the plants) is volatile and could be transferred through the vapor phase within the closed bud Alternatively, specific translocation of jasmomc acid or its derivatives in the vascular system may be involved
A second component of male-stenlity in the triple mutant is the failure of the anther locules to dehisce correctly In some male-stenle mutants,
the pollen is fully viable and the failure of pollination and fertilization is attributable solely to the fact that anther dehiscence does not occur (Dawson et al , Can J Bot. 71 629-638, 1993) Other male-stenle plants, m which pollen
death results from selective destruction of the tapeturn, are able to undergo anther dehiscence normally (Manam et al , Nature 347 737-741, 1990) These examples indicate that the maturation of viable pollen is not directly linked to the complex processes that lead to dehiscence of the anther locules and release of the pollen (Keyzer, New Phytol 105 487-498, 1987) In addition, the possibility
that jasmomc acid affects dehiscence only through its actions in pollen maturation can not be ruled out However, in light of the findings of Mariaru et al (Nature
347 737-741, 1990) and Dawson et al (Can J Bot 71 629-638, 1993), it is more likely that jasmomc acid performs (at least) two separate signalling functions dunng flower development, first, to ensure the maturation of viable
pollen, and second, to orchestrate the changes in cell wall structure of the stomium and in the cellular water relations within the endothecium that result m successful dehiscence of the anthers (Stanley and Linskens, Pollen Biology,
Biochemistry and Management, New York, Springer-Verlag, 1974).
EXAMPLE 7. Proximity Requirements in the Administration of Femlitv-30 Restoring Agents
The observation that a-linolemc acid could restore fertility to .lowers without penetrating the flower bud to increase the proportion of a-lmolemc acid in the anthers to measurable levels (detection limit approximately 0 1 % of total
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fatty acids) suggests that jasmomc acid may be produced in other flower organs on mutant plants and then diffuse or be transported to anthers
When jasmonate was painted on the flower buds of fadi-2 fad7-2 fad8 tnple-mutant plants, consistent seed set was observed as desenbed above 5 However, when the application of jasmonic acid was stopped, further flowers on the plants were sterile even though these sterile flowers developed in close proximity to the jasmonate-treated buds that produced fertile flowers Furthermore, neither wild-type plants nor tnple-mutant plants that were rendered fertile through the application of exogenous jasmonic acid induced any seed set in 10 untreated tnple-mutant plants growing in close proximity to them
These observations indicate that the femlity restonng effects of jasmomc acid and related compounds requires direct or close contact of the jasmomc acid or related compound to the individual flowers that are to produce fertile pollen Such a proximity requirement is likely to be an asset m the use of IS conditionally male-fertile plants for hybnd breeding because it is unlikely that fertility will be inadvertently restored by, for example, the presence of male-fertile plants grown close to plants of the male-stenle line
EXAMPLE 8 A Second Male-Sterile Line in Which Fertility is Restored bv 20 Jasmonic Acid
In the catalogs of the Arabidopsis Biological Resource Center at Ohio
State University (Columbus, Ohio 43210, USA) and the National Arabidopsis
Stock Center at the University of Nottingham (Nottingham, UK), a total of 24
unrelated lines of Arabidopsis (ecotypes Wassilewskija and Columbia) are listed
as lines that segregate male-stenle individuals CS2321, CS2322, CS2324,
CS2811, CS2326, CS2328, CS2329, CS2331, CS2332, CS2336, CS2337,
CS2338, CS2814, CS2340, CS2341, CS2342, CS2343, CS2347, CS2349,
CS2350, CS2351, CS2352, NW75, N393
Sixteen seeds from each of these lines were germinated and the
resulting plants grown as described above for approximately four weeks At this stage, fertile segregants in each line could be identified by the presence of enlarged, fertile siliques Conversely, stenle segregants lacked enlarged siliques
Sixteen of the lines yielded stenle segregants and unopened flower buds on these
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stenle plants were painted either with water or with an aqueous solution containing 0 1 /imol/ml of jasmomc acid Of the lines tested in this way, only the sterile segregants of line CS2338 remained consistently sterile after treatment of buds with water and produced seeds when buds were painted with jasmonic 5 acid solution This result was subsequently confirmed on other sterile plants of this line The seeds produced on sterile plants following treatment with jasmomc acid consistently gave rise to progeny plants that were sterile, indicating that the seeds could not have resulted by fertilization by pollen from other plants In subsequent experiments, flower buds on stenle plants of line 10 CS2338 were treated with solutions of bestatm made up in 10 mM potassium phosphate buffer (pH 6 3) The flower buds on twelve stems were treated with a solution containing 1 0 mM bestatm each day *or three days These stems subsequently produced more than 100 siliques from the treated buds, presumably because bestatm treatment led to the production of viable pollen The restoration IS of fertility resulting from application of bestatm was quantitatively similar to that obtained using 0 1 /unol/ml of jasmomc acid or methyl jasmonate A solution containing 100 (M bestatm was approximately half as efficacious, while a 10 mM potassium phosphate buffer solution, pH 6 3 (without bestatm) was approximately one-tenth as efficacious, as 1 mM bestatm in restoring fertility 20 Treatment of fad3-2 fad7-2fad8 plants with 1 mM bestatm did not result in production of any seeds These results indicate that bestatm is able to restore fertility in at least some jasmonate-dependent conditionally-fertile plant lines
The male-sterile phenotype of line CS2338 included morphological features m common with that of the fad3-2 fad7-2 fad8 triple mutant Floral 25 organs developed normally and the anther locules were not dehisced in flowers that had developed for two days after the petals appeared at the tip of the bud When the anther locules were disrupted, the pollen that was released was not capable of fertilizing emasculated wild-type flowers These results indicated that line CS2338 was indeed segregating for a male-stenle trait 30 Seed was collected separately from several individual fertile segregants of line CS2338 to form a senes of sublines Seeds from each subline were germinated and the plants grown to maturity Some of the sublines
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produced 100% fertile plants, while some produced both fertile and stenle plants in ratios that approximated 3 fertile 1 stenle These results indicated that the male-stenle trait in line CS2338 resulted from a recessive allele of a single nuclear mutation
The fatty acid composition of leaves, whole flowers and anthers from a sterile plant of line CS2338 and a wild-type control (Arabidopsis ecotype Wassilewskija) were determined by gas chromatography analysis essentially as described by Miquel and Browse (J Biol Chem 267 1502-1509, 1992) The mutant hne showed no substantial deficiency in a-lmolenate in any of the tissues 10 analyzed Therefore, sterility in the mutant did not result primarily from a deficiency of a-linolemc acid, as appears to be the case in the fad3-2 fad.7-2 fad8 triple mutant line
Furthermore, attempts to make male-stenle plants of the CS2338 line fertile by applying the sodium soap of a-linolenic acid did not result m any seed 15 set Clearly, the mutation in line CS2338 affected a step m jasmonate synthesis or action that is independent of the supply of a-linolemc acid substrate Flower buds from sterile plants of line CS2338 and wild-type Arabidopsis were harvested and assayed for jasmonate content using the method of Albrecht et al (Planta 191 86-94, 1993) The concentrations of jasmonate m stenle and wild-type 20 (fertile) flowers were comparable (averaging 193 and 205 pmol/g fresh weight, respectively), suggesting that the defect in stenle plants of line CS2338 either affects the availability of endogenous jasmonate (or related compounds) to its receptor in the signalling pathway or involves a mutation in the receptor system that necessitates the application of exogenous jasmonate in order to activate the 25 signalling system and render the plants fertile
This invention has been detailed both by example and by direct descnption These Examples, while indicating preferred embodiments of the invention, are illustrative only From the descnption given above, one skilled in the art can ascertain the essential charactenstics of this invention and, without 30 departing from the spirit and scope thereof, can make vanous changes and modifications of the invention, which are to be included within the scope of the invention
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