Patent Grant
9516824
Sexual reproduction in multi-cellular organisms entails generation of meiotically competent germ cells within a somatic body. Developmental mechanisms responsible vary among taxa, however, most animals exhibit continuous production from stem cells specified during embryogenesis. In contrast, angiosperms are strictly vegetative until intrinsic and environmental cues trigger flowering. Within anther and carpel primordia, indeterminate floral progenitor cells differentiate as pre-meiotic archesporial (AR) cells and somatic parietal cells, but the morphogenetic mechanisms responsible remain unclear. The nature of the somatic to germinal switch, and the degree to which it is under developmental or physiological control, has until now been a botanical mystery.
A method of altering the number of archesporial cells in a developing anther of a plant is provided. In certain embodiments, the method comprises exposing the anther to redox-modulatory conditions prior to differentiation of germline cells in the anther, thereby changing the redox potential of cells in the anther and altering the number of archesporial cells in the anther. This method may be employed to increase or decrease the number of archesporial cells in a developing anther
In one embodiment the treatment may comprise exposing the anther to hypoxic conditions or to a reducing agent at a concentration that lowers the amount of reactive oxygen species in the cells of the anther, thereby lowering the amount of reactive oxygen species in the cells and increasing the number of archesporial cells. Increasing the number of archesporial cells in the anther may result in a plant having larger anther size and/or higher pollen production, relative to a control plant that has not been exposed to hypoxic conditions or to a reducing agent. In one case, this method may be done by placing the anther in an environment that contains less than 1% oxygen, e.g., in a gas containing at least 99% nitrogen. Alternatively the developing tassel can be immersed in redox-modulating chemical solutions by injecting fluid into the airspace surrounding the immature tassel at the stage just prior to or during archesporial cell formation.
In another embodiment, the exposing may comprise contacting the anther with an oxidizing agent (such as pure oxygen gas or chemicals) at a concentration that increases the amount of reactive oxygen species in the cells, thereby increasing the amount of reactive oxygen species in the cells and decreasing the number of archesporial cells. In certain cases, decreasing the number of archesporial cells in the anther results in a plant having smaller anther size and/or lower pollen production than a control plant that has not been subjected to the applying. In other cases, decreasing the number of archesporial cells may result in a male sterile plant. In particular cases, the oxidizing agent may be a peroxide, although any other suitable oxidizing agent may be applied.
A developing anther may be exposed to redox-modulatory conditions in a variety of different ways. For example, in one embodiment, the exposing may comprise exposing the developing anther to a gas. In another embodiment, the exposing may comprise contacting the developing anther with a liquid or gel that comprises a redox-modulatory compound, e.g., by spraying the anther or contacting the anther with a droplet. In this embodiment, the redox-modulatory compound may be dissolved in the liquid or gel, or the redox-modulatory compound is in or on a particle that is present in the liquid or gel. In particular cases, the particle may provide for extended release of the redox-modulatory compound over a period of, e.g., 1 to 5 days. In other embodiments, the applying may comprise placing a solid form of a redox-modulatory compound on the developing anther.
The method summarized above finds use in a variety of applications, such as, e.g., to increase anther size and/or increase the number of pollen produced by a plant or to make plants with a decrease in anther size and/or a decreased number of pollen. In one example, the method may be used to make a male sterile plant. This method may comprise: exposing the developing anthers in an anther of a plant, prior to differentiation of germline cells, to an oxidizing agent at a concentration that increases the amount of reactive oxygen species in cells in the anthers, thereby increasing the amount of reactive oxygen species in the cells and decreasing the number of archesporial cells in the anthers; and cultivating the plant to produce a male sterile plant. This method may further comprise crossing the male sterile plant with a another plant to produce an hybrid plant, e.g., a plant that has hybrid vigor relative to its parents. This method, as will be discussed below has significant utility in the production of hybrid monocots, e.g., corn and rice.
Also provided is a plant comprising a pre-meiotic anther having a non-heritable increase in the number of archesporial cells, relative to a plant of the same germplasm grown in air with out an application of an oxidizing agent.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.
All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.
The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
As used herein, the term “archesporial cell” refers to an cell in an anther primordium from which the microsporocytes of a flowering plant develop. Archesporial cells from a variety of different model monocot and dicot species are described in, e.g., Raghavan (J. Cell Sci. 1989 92:217-2; rice); Sheridan et al (Genetics. 1996 142:1009-20; maize), Sheridan et al (Genetics 1999 153: 933-41; maize); Feng et al (Development 2010 137:2409-1; Arabidopsis); Ma et al (Plant J. 2007 50:637-48; maize) and Cnudde et al (Chromosome Res. 2006 14: 919-32; petunia). These references are incorporated by reference for a description of those cells.
As used herein, the term “prior to differentiation of germline cells” refers to a stage in anther development after the stamen primordia have been initiated from a meristem and prior to the production of meiotically competent germ cells within a somatic body. This stage is considered to be early in anther development.
As used herein, the term “redox-modulatory conditions” refers to the conditions that increase the amount of reactive oxygen species in a cell relative to the same type of cell that is grown under equivalent conditions in air, i.e., the earth's atmosphere, at ground level. In other words, under this definition, air (which is composed of approximately 79% nitrogen, 20% oxygen, and 1% other gases) is not considered a redox-modulatory condition. However, air may contain components, e.g., nitrogen and oxygen, which, if they are applied at a concentration that is different their concentration in air (e.g., less then 1-% oxygen, at least 90% nitrogen, at least 30% oxygen or less than 70% nitrogen, etc.), can be considered redox-modulatory because they can increase or decrease the amount of reactive oxygen species in a cell. Redox modulator conditions can be created by exposing a developing anther to hypoxic conditions (e.g., an environment containing less than 1% oxygen), by contacting a developing anther with a redox-modulatory compound, e.g., a reducing agent or oxidizing agent, at a concentration that alters the amount of reactive oxygen species in the cells of the anther.
As used herein, the term “reducing agent” refers to a compound that donates an electron to another species within a cell, thereby reducing the oxidation state of a cell.
As used herein, the term “oxidizing agent” refers to a compound that removes electrons from another reaction in a cell, thereby increasing the oxidation state of a cell. Oxygen is a type of oxidizing agent. However, as noted above, if oxygen is used as an oxidizing agent, it must be applied at an amount that is greater than its concentration in the earth's atmosphere.
As noted above, a method of altering the number of archesporial cells in a developing anther of a plant is provided. In general terms, the method comprises exposing the anther to redox-modulatory conditions prior to differentiation of germinal cells in the anther, thereby changing the redox potential in precursor L2-d cells (layer 2 derived cells tracing back to the floral meristem) and altering the number of archesporial cells in the anther. Because the archesporial cells develop into microsporangia cells, an increase in the number of archesporial cells leads to larger anthers and/or more pollen, and a decrease in the number of archesporial cells leads to smaller anthers and/or less pollen, or male sterility.
In certain embodiments an anther may be contacted with a reducing agent at concentration that lowers the amount of reactive oxygen species in the cells of the anther, thereby lowering the amount of reactive oxygen species in the cells and increasing the number of archesporial cells. The same effect may be obtained by subject the anther to hypoxic conditions, using, e.g., an inert gas at a concentration that makes the cells hypoxic, which lowers the amount of oxygen, and hence produces lower amounts of reactive oxygen species in the cell. In these embodiments, the concentration of oxygen in the inert gas may be less than 15%, less than 10%, less than 5%, or less then 1%. In particular cases, the gas may be composed of a single element (e.g., N2 gas), although the gas may be a mixture of elements in certain cases. For example, if N2 gas is used, then the N2 may be present in the gas at a concentration that is greater than 80%, e.g., at least 85%, at least 90% or at least 95%, thereby creating hypoxic conditions. Several reducing agents are known in the art and include Na, Cr, Cu and Cl−. Common reducing agents contain potassium, calcium, barium, sodium and magnesium, and also compounds that contain an H− ion, including NaH, LiH, LiAlH4 and CaH2. Reducing agents that are suitable for use in this embodiment of the method include lithium aluminium hydride (LiAlH4), sodium amalgam, sodium borohydride (NaBH4), compounds containing the Sn2+ ion, such as tin(II) chloride, sulfite compounds, hydrazine (Wolff-Kishner reduction), zinc-mercury amalgam (Zn(Hg)) (Clemmensen reduction), diisobutylaluminum hydride (DIBAH), lindlar catalyst, oxalic acid (C2H2O4), formic acid (HCOOH), ascorbic acid (C6H8O6), phosphites, hypophosphites, phosphorous acid, dithiothreitol (DTT) and several compounds containing Fe2+, such as iron(II) sulfate. Lowering the amount of reactive oxygen species increases the number of archesporial cells in the anther results in a plant having larger anther size and/or higher pollen production, relative to a control plant that has not been subjected to the applying, i.e., a control plant that has not been exposed to the reducing agent or to a gas or hypoxic conditions. An increase in the number of archesporial cells is desirable for production of products that are made from pollen and/or anthers. For example, an or decrease increase in the number of archesporial cells is desirable in saffron production (which is produced from carpels), for the production of pollen that can be used in dietary supplements, for the production of medicinal compounds, or to produce allergen for inoculations and testing.
In other embodiments, an anther may be contacted with an oxidizing agent at a concentration that increases the amount of reactive oxygen species in cells of the anther. This decreases the number of archesporial cells. Several oxidizing agents are known in the art and include oxygen (O2), ozone (O3), peroxide such as hydrogen peroxide (H2O2) and inorganic peroxides, fluorine (F2), chlorine (Cl2), and other halogens, nitric acid (HNO3) and other nitrates, sulfuric acid (H2SO4), persulfuric acids (H2SO5 and H2SO8), chlorite, chlorate, perchlorate, and other analogous halogen compounds, hypochlorite and other hypohalite compounds, including bleach (NaClO), hexavalent chromium compounds such as chromic and dichromic acids and chromium trioxide, pyridinium chlorochromate (PCC), and chromate/dichromate compounds, permanganate compounds, sodium perborate, nitrous oxide (N2O). Increasing the amount of reactive oxygen species decreases the number of archesporial cells in the anther results in a plant having smaller anther size and/or lower pollen production or male sterility, relative to a control plant that has not been subjected to the applying, i.e., a control plant that has not been exposed to the oxidizing agent. Again, if oxygen is used as an oxidizing agent, it is used at a concentration that provides an increased concentration of oxygen in the cells relative to cells in a control plant grown in the earth's atmosphere, which leads to more reactive oxygen species in the cells of the developing anther.
A developing anther of a plant may be exposed to redox-modulatory conditions in a variety of different ways. For example, in one embodiment, the exposing may comprise exposing the developing anther to a gas. In this embodiment, at least part of a plant (e.g., the entire plant or an inflorescence) may be enclosed in an enclosure (e.g., in a bag or, if many plants are being treated, in a tent) and a gas (e.g., nitrogen, oxygen, or another gas) may be added to the interior of the enclosure, thereby increasing or decreasing the oxygen concentration in the enclosure relative to the outside air. In another embodiment, the exposing may comprise contacting the developing anther with a liquid (e.g., by spraying the liquid) or gel that contains a redox-modulatory compound. Alternatively the developing tassel can be immersed in redox-modulating chemical solutions, e.g., by dipping or injecting fluid into the airspace surrounding the immature tassel at the stage just prior to or during archesporial cell formation.
The redox-modulatory compound may be dissolved in a medium that is applied to the anther, or it may be present in or on a particle that is present in the medium that is applied to the anther. In another embodiment, the exposing may by placing a solid form of the redox-modulatory compound on the developing anther, e.g., a crystal or particle containing the redox-modulatory compound. As would be apparent, the redox-modulatory compound should be applied so that decrease or increase in reactive oxygen species occurs at an appropriate time in anther development, i.e., after anther primordia have formed but prior to differentiation of germinal cells. The optimal time frame for altering the number or presence of germinal cells is to initiate treatments just before or at the onset of germ cell specification. This period occurs in 100-200 micrometer length maize anthers (Kelliher & Walbot 2012). In other species, anther size is smaller at the comparable stage: stage 5 of Arabidopsis floral development (Smyth, D. R., Bowman, J. L., and Meyerowitz, E. M. (1990). Early flower development in Arabidopsis. Plant Cell 2, 755-767) or tobacco (Nicotiana tabacum Goldberg, R. B., Beals, T. P., and Sanders, P. M. (1993). Anther development: Basic principles and practical applications. Plant Cell 5, 1217-1229 In rice (Itoh, J-I. et al. 2005. Rice plant development: from zygote to spikelet. Plant & Cell Physiol. 46: 23-47) the correct stage is designated as stage Sp6 formation of stamen primordia extending into stage SP7. In lily, the stage is prior to the 1 mm length anther (Wang et al. 1992. Patterns of protein accumulation in developing anthers of Lilium longiglorum correlate with histological events. Amer. J. Botany 79: 118-127. In Petunia hybrida, the appropriate stage is Stage 1 (Gillman et al. 2009, chapter 6 in Petunia: Evolutionary, Developmental and Physiological Genetics, ed. T. Gerats and J. Strommer). Information on early anther development is available for additional species in D'Arcy, W. G. and R. C. Keating's 1996 book The anther: form, function, and phylogeny. This period of time may vary from plant to plant. However, because similar morphological events occur in all plants, the appropriate time period for application of the redox-modulatory compound may be readily determined. The optimal period may be experimentally determined. In particular cases, the entire plant may be exposed to the redox-modulatory conditions. In other embodiments, only an inflorescence (e.g., the tassel of a maize plant or other monocot) may be exposed to the redox-modulatory conditions. In particular cases, the anther may be exposed to the redox-modulatory conditions more than once. In certain cases, an exposure may be for extended for a period of time, e.g., 6 hr to 1 week, or 1 to 5 days, as desired.
A redox-modulatory compound can be applied to a plant either by itself or as a formulation that also contains an agronomically acceptable carrier and, optionally, other active ingredients. By “agronomically acceptable carrier” is meant any liquid or solid substance that can be used to dissolve, disperse, or diffuse a redox-modulatory compound without impairing the effectiveness of the compound and which by itself has no significant detrimental effect on the soil, equipment, crops, or agronomic environment. Such compositions include liquid or solid formulations or solutions, including wettable powders, emulsifiable concentrates, dusts, granules, pellets, aerosols, flowable emulsion concentrates, suspensions, and solutions, which may be prepared according to any suitable method. A formulation containing a redox-modulatory compound can be diluted with an agronomically suitable liquid or solid carrier. Such compositions can also include one or more agronomically acceptable adjuvants such as anionic, cationic, or nonionic surface-active agents (wetting agents, spreading agents, dispersing agents, suspending agents, and emulsifying agents), conditioning agents, sticking agents, adhesives, etc. Examples of useful adjuvants can be found in “Detergents and Emulsifier's Annual” (John W. McCutcheon, Inc.).
A redox-modulatory compound may in certain cases be administered as a liquid or wettable powder, containing as a conditioning agent one or more surface-active agents in amounts sufficient to render the redox-modulatory compound readily dispersible in water or in oil. The incorporation of a surface-active agent into the compound can enhance its efficacy. Suitable wetting agents include but are not limited to alkyl benzene and alkyl naphthalene sulfonates, sulfonated fatty alcohols, amines or acid amides, long chain acid esters of sodium isothionate, esters of sodium sulfonsuccinate, sulfated or sulfonated fatty acid esters, petroleum sulfonates, sulfonated vegetable oils, ditertiary acetylenic glycols, polyoxyethylene derivatives or alkylphenyls (particularly isooctylphenol and nonylphenol) and polyoxyethylene derivatives of the nono-higher fatty acid esters of hexitol anhydrides (e.g., sorbitan). Surfactants include, but are not limited to, the dihexyl ester of sodium sulfonsuccinic acid, POE 20 sorbitan monolaurate, and octylphenoxy polyethoxy ethanol. Wettable powders or dispersable granules are water-dispersible compositions containing one or more active ingredients, an inert solid extender, and one or more wetting and dispersing agents. The inert solid extenders may be of mineral origin such as the natural clays, diatomaceous earth, salts and synthetic minerals, derived from silica and the like. Examples of such extenders include kaolinites, attapulgite clay, salts and synthetic magnesium silicate.
A redox-modulatory compound can also be dissolved in any suitable solvent, including but not limited to one or a mixture of the following: water, alcohols, ketones, aromatic hydrocarbons, halogenated hydrocarbons, dimethylformamide, dioxane, and dimethylsulfoxide. The concentration of the redox-modulatory compound in the resulting solution may be in the range of about 2% to about 98% by weight, e.g., from about 20% to about 75% by weight.
Wettable powders suitable for spraying are mixtures of a redox-modulatory compound, a finely divided solid (such as a clay, an organic silicate or carbonate, or a silica gel), and a wetting agent, sticking agent, and/or dispersing agent. The concentration of the active ingredient(s) in such powders is generally between about 20% and about 98% by weight, e.g., between about 40% and about 75% by weight. A dispersion agent is optionally present in a concentration of about 0.5% to about 3% by weight of the composition. A wetting agent may constitute from about 0.1% to about 5% by weight of the composition.
A dust containing a redox-modulatory compound may also be employed, e.g., one made from a finely divided inert organic or inorganic solids such as a botanical flour, farina, diatomite, silicas, silicates, carbonates, and clays. One method for preparing a dust is to dilute a wettable powder with a finely divided carrier. A dust concentrate containing from about 20% to about 80% of the redox-modulatory compound can be diluted to a final concentration of about 1% to about 10% by weight of the dust.
Particulate (e.g., granular) formulations can be prepared by impregnating the active ingredient(s) into a solid material. A solution of a formulation in a volatile organic solvent is sprayed or mixed with the granular solid and the solvent may be removed by evaporation. The granular material can have any suitable size, e.g., 11 to about 60 mesh. The redox-modulatory compound may represents about 2% to about 15% by weight of the formulation. Alternatively, the formulation can be incorporated into controlled-release particulate formulations by standard methods, e.g., by encapsulation by interfacial polymerization and coacervation; dissolving the active ingredient in a solution together with a polymer followed by solvent evaporation; by mixing the active ingredient with a wax or polymer (by mixing dry ingredients followed by melting the mixture or by mixing the active ingredient with a molten wax or polymer, followed by solidification of the mixture), then producing particles of the mixture by prilling, milling, extrusion, spray chilling, etc. The active ingredient generally represents between about 5% and about 50% of such a controlled-release formulation.
If a salt is employed, the salt may be formulated and applied as an aqueous solution at a concentration of between about 0.05% to about 50% by weight, e.g., from about 0.1% and about 10% by weight and applied to plants in this form. Such solutions can be prepared as concentrates that are diluted with an aqueous solvent or other appropriate solvent to the desired concentration for use. Such solutions optionally include a surface active agent and/or one or more auxiliary materials to increase the activity of the active ingredient, such as glycerin, methylethylcellulose, hydroxyethyl cellulose, polyoxyethylenesorbitan monooleate, polypropylene glycol, polyacrylic acid, polyethylene sodium malate, or polyethylene oxide, etc.
The formulation described above of the invention can be applied by conventional method, including, but not limited to mechanical application and manual application. For low-volume applications a solution of the compound may be used. In one embodiment, each inflorescence is individually sprayed with a controlled release formulation such that the redox-modulatory compound is released over a period of time, e.g., over 1-5 days, thereby changing the redox potential of cells at the appropriate period of development. An anther may also receive multiple doses of the compound, if necessary. The optimum formulation, volume, concentration, application rate, timing of application (including stage of plant development), and method of application will depend on a variety of factors such as plant type, soil type, fertility, environmental factors, etc.
As noted above, the subject method may be used to induce male sterility in plants in a method that comprises applying a redox-modulatory compound to an anther of a plant prior to differentiation of germline cells in the anther, thereby changing the redox potential of cells and altering the number of archesporial cells in the anther; and cultivating the plant to produce a male sterile plant. The plants made by this method generally comprise a pre-meiotic anther having a non-heritable increase in the number of archesporial cells, relative to plant of the same germplasm grown in air without an application of an oxidizing agent. The method may further comprise crossing the male sterile plant with another plant to produce a hybrid plant. The ability to produce male sterile plants is particularly valuable for the production of seed that give rise to hybrid plants that have greater vigor than its inbred parents.
For hybrid seed production in the field, the two parent strains to be crossed may be planted in alternate sections, rows, or groups of rows. The female parent is treated as described above in order to render the female parent male sterile. Pollen from the male (untreated) parent then fertilizes the female parent, either by means of human intervention or by a natural process, such as wind-borne pollination. The seed produced by the female parent is an F1 hybrid, which is then collected by any suitable means. Plants can be crossed by either natural or mechanical techniques. Natural pollination occurs when pollen is transported by gravity, wind, pollinating insects or animals or other natural vectors from the male reproductive parts of a flower to the receptive portions of the flower. In monoecious crops, such as maize, the male and female flower parts are positioned at different locations on the same plant. In dioecious plants, there are separate male and female plants.
In one embodiment, seed produced is a first generation seed capable of being grown into an F1 hybrid plant, where both the first and second parents of the hybrid are inbred plants. In another embodiment, one or both of the first and second parent plants are themselves hybrids. In one embodiment, this method comprises: (a) planting seeds of a first and a second parent plant; (b) growing the first and second parent plants; (c) treating at least the first plant so as to make it male sterile, as described above; and (d) cross-pollinating the treated plant with pollen from the second parent plant. Both parental plants may be allowed to continue to grow until maturity or the male rows may be destroyed after flowering is complete. Therefore, in certain embodiments, this method may include the next step of: (e) harvesting seeds resulting from the cross-pollinating. Only seeds from the female parental plants are generally harvested to obtain outcrossed seeds. The collected seed represents a valuable commercial product which can be sold to farmers, processed, or employed in further breeding programs.
The method described above and exemplified below may be readily adapted for use in the production of hybrid dicotyledonous crops (including, but not limited to, sugar beet, sugarcane, potato, sweet potato, lettuce cabbage, tea, radish, turnips, garlic and onion) and monocotyledonous crops, including, but not limited to, graminaceous crops such as wheat, barley, maize, rice, sorghum, millet, oats, rye, triticale, turf and forage grasses, etc.
In order to further illustrate the present invention, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope.
Maize anthers consist of four pollen-containing sacs, called lobes, surrounding a central vasculature (VT); together these form a butterfly shape in transverse-section. Anther development begins with three stamen primordia initiating from a floret meristem (
Based on microscopic evaluation, it was believed that archesporial and parietal cells arise simultaneously from an asymmetric cell division (ACD) of an enlarged hypodermal cell at the apex of each locular arch when locules contain few Layer 2-derived cells (L2-d cells, tracing back to the second meristematic layer). Because rigid walls prohibit plant cell movement, these two cell types establish the lineages ultimately resulting in anther functional anatomy with further differentiation requiring continuing positional cues. Despite widespread invocation of lineage, hypodermal cells and ACD have not been rigorously documented. At the budding stage, there are 15-20 haphazardly arranged globular L2-d cells in a maize locule (
Fertile W23 inbred anthers were stained with propidium iodide and imaged in Z-stack using a Leica SP5 confocal microscope (6). The ontogeny of locular cell types was cataloged over ˜2.5 days as anthers grew in length from 100 to 300 μm. From reconstructions summarized in longitudinal perspective (
For each AR birth, the pluripotent parent cell was identified by the thin wall shared with a sister L2-d. In W23 most progenitors were located at the keystone position viewed transversely (63%, 67/106), as posited in the lineage model, 21% were lateral ( 22/106), and 16% were basal ( 17/106) (
AR specification is a dynamic process that initiates centrally and proceeds towards the base and tip, ending by ˜220 μm (
For comparison, morphometric analysis was performed on inbred A619, which has a slower flowering progression and fewer flowers than W23. Despite these differences, the developmental stages were identical. As in W23, AR specification lasted from 120 to 220 μm with morphological differentiation apparent at 160 μm. Notably, A619 locules averaged twice as many AR cells as W23 (
At 300 μm multiple archesporial cells1 (mac1) locules contain only a single somatic layer surrounding excess AR (19). The mutant was introgressed into W23 and compared to fertile siblings. From the onset (<120 μm) and subsequently, locules had extra L2-d cells (
Two Arabidopsis mutants are similar to mac1: the LRR receptor kinase EXS/EMS1 and its putative secreted ligand TPD1, a homolog of rice OsTDL1A. These molecules are proposed to define a signaling module responsible for tapetal specification. Alternatively mutants in this module may suffer from a failure to thrive syndrome of TA initials because unlike mac1, tpd1 and ems1 mutants typically form EN and SPL but ML and TA specification is faulty, except in the C24 background exs phenocopies mac1. MAC1 contains a predicted cleavable signal peptide.
The single layer mac1 soma has a cell census similar to the sum of EN and SPL in fertile siblings; mac1 somatic cells are smaller than either SPL or EN reflecting increased anticlinal division to sustain anther elongation (
AR proliferation dramatically increased: 30% of mac1 AR cells were EdU+ compared to 12% in fertile (
Mac1 expression is low in tassel and anther primordia; there is a burst of expression in ˜150 μm anthers, when the first AR cells morphologically differentiate with increases at subsequent stages. Mac1 is also significantly enriched in laser-microdissected AR cells at both the 300 μm and 700 μm stages compared to the EN, SPL, ML, and TA layers (
Anatomically normal msca1 anthers contain none of the correct cell types. During locule budding msca1 anthers are identical to fertile, however, globular progenitor cells surrounded by L2-d continue to proliferate then differentiate as columnar vasculature (
Reactive oxygen species (ROS) affect many plant developmental processes, including root hair elongation, leaf growth, and root transition zone placement. During AR specification, the tassel is tightly encased within a whorl of not yet photosynthetic leaves. As a sink tissue undergoing rapid growth, the tassel and surrounding leaves have high metabolic demand, and we reasoned oxygen could be depleted in the small air space (˜1 cm3) between the tassel and innermost leaf. To determine oxygen concentration, we inserted a needle-borne probe at several developmental stages (
N2 or O2 gas was administered through hoses threaded into the leaf whorl (
Compared to untreated fertile anthers, all three N2 protocols resulted in early specification and excess AR cells, phenocopying the first component of mac1 development (
In contrast, the hose O2 treatment repressed AR specification: central AR counts were far lower than N2 after 48 hour treatment (
Cellular redox was perturbed chemically by injecting 1 mL of 1 mM H2O2 or 10 mM KI (a peroxide scavenger). While KI did not alter total L2 or peripheral counts, H2O2 treatment greatly reduced these cell numbers (
Collectively, these treatments highlight the key role played by redox in the specification of AR cells in that the L2-d cells are poised for a redox-dependent signal relayed through MSCA1 to establish AR fate. In this developmental context, hypoxia increases cell proliferation, placing more L2-d cells in central positions earlier where AR fate specification normally starts; conversely treatments that increased oxygen/ROS suppressed AR specification and hence also delayed somatic niche formation.
Ectopic AR were identified based on characteristic morphology and ability to direct periclinal divisions locally (
In total, 4.3% of 1490 anthers imaged had ectopic AR cells; 3.0% and 6.2% in oxidizing and reducing treatments, respectively (Table 2 and 3). AR location was biased depending on treatment. In oxidizing treatments, 70% of ectopic AR were near the VT (
Gas and chemical treatments caused ectopic AR cells in mac1 anthers, but there was no subsequent stimulation of periclinal division in neighboring L2-d, confirming that somatic niche formation requires MAC1 (
Many thioredoxins are required during floral organ development, suggesting excessive ROS cause sterility. Genes that lower ROS and support reducing capacity are expressed in young anthers by microarray analysis (Table 4). Many of these are specifically enriched in laser microdissected AR at later stages (Table 5) and absent in msca1 (Table 6), suggesting they are important in the germline. Genes involved in energy generation that bypass the mitochondrial electron transport chain, a major source of ROS (36) are highly represented. Five of seven glyoxylate shunt enzymes are enriched in AR cells (Table 5) and two are missing in msca1, suggesting an increased capacity in this peroxisomal/cytoplasmic process in AR cells. These alternative pathways avoid ROS production, facilitating maintenance of cellular hypoxia.
AR specification activates cascades of gene expression in hormone pathways—up-regulation of enzymes for making growth regulators ethylene, gibberellins, and cytokinin and jasmonic acid controlling selective abortion of female floral parts in maize tassel florets. Transcripts for controlling cytokinin and jasmonic acid production are absent in msca1 (Table 6). Conversely, abscisic-aldehyde oxidase, which produces the hormone abscisic acid and H2O2, is upregulated in msca1 anthers and missing from AR cells (data not shown).
In conclusion, the analysis described above has debunked the lineage model through discovery that multiclonal AR arise within a field of pluripotent cells all expressing the MAC1 proliferation regulator. It is proposed that the central locular cells—those with only L2-d neighbors where locules are widest—achieve a hypoxic threshold to trigger MSCA1 glutaredoxin-mediated activation of the AR specification pathway. MAC1 production may be rapidly elevated in AR, which become signaling centers to repress their own proliferation and to activate neighboring L2-d cells to conduct a single periclinal cell division to establish the EN and SPL. These events proceed from the center towards the anther base and tip, resulting within 40 hours in locules with a column of central AR cells encircled by two somatic rings (
The capacity to differentiate as an AR cell is not restricted to central L2-d cells. When a more reducing environment is imposed, subepidermal cells can become AR; in an oxidizing environment, internal connective cells adjacent to the vasculature can differentiate as AR. These observations reinforce the conclusion that AR differentiation is an emergent property dependent on physiological conditions and not the consequence of lineage or unique cell division patterns. Our results illustrate the inherent plasticity in plant development and capacity to reprogram cellular fate. In contrast to animals, plant germinal cells arise first and organize their somatic support tissues and can mature to functional meiocytes in the absence of normal soma.
The tables discussed above are described in more detail below.
Table 1. Pre-meiotic (1000 μm) AR-enriched transcripts in fertile and mac1. Laser microdissected AR cells from mutant and fertile sibling were compared on microaffay in duplicate (dye swap). Expression of 297 genes found to be enriched in AR cells was nearly identical (96.4% similar) between the two sample types according to ON/OFF categorization. 3.3% of transcripts were present and low in fertile but absent in mac1.
Table 2. Ectopic AR formation in reducing treatments that increase hypoxia or lower H2O2. Ectopic AR cells were defined by their morphological similarities with normal AR cells combined with non-locular location. The first two rows give the treatment type and genotype, and the next two rows give the general effects of the treatments on AR counts and SPL/EN progression as presented in
Table 3. Ectopic AR formation in oxidizing treatments that increase oxygen and/or H2O2. Ectopic AR formation in oxidizing treatments was highly biased for the internal tissues. The organization of the table is the same as in Table 2.
Table 4. Ninety-eight transcripts found in early fertile anthers associated with redox regulation, metabolism, alternative energy metabolism, and hormone biosynthesis or signaling. A number of genes associated with the glyoxylate cycle (e.g. malate dehydrogenase, succinate dehydrogenase, pyruvate dehydrogenase) and ROS management (e.g. superoxide dismutase, glutathione S transferase, thioredoxin, and glutaredoxin-like) are highly expressed. These probes were chosen for inclusion here based on slight enrichment in mac1 mutant anthers (contains extra AR cells) to focus on genes that might be enriched or specifically expressed in AR cells.
Table 5. Pre-meiotic (650-750 μm) AR-enriched transcripts. Laser microdissected AR cells were compared in duplicate (dye swap) to whole anthers (WA) from the same tassel at the same stage. Genes that were expressed two-fold higher in AR versus WA have a log 2ratio>0.58 (p<0.05). Some genes just below the cutoff are listed because of their importance to either alternative metabolism or ROS handling. Along with many genes that scavenge ROS or manage reducing power (NADH enzymes and glutaredoxins, for example), 5 of the 7 genes of the glyoxylate cycle are enriched, suggesting AR cells are specifically using this alternative pathway to generate ATP without the side effect of endogenous ROS production.
Table 6
Genes down-regulated in early msca1 versus fertile anthers. RNA extracted from fertile and msca1 whole anthers at the 200 m stage (just as the AR fate specification period is ending) were compared by microarray in duplicate (dye swap). Genes that were expressed two-fold lower in msca1 versus fertile have a log 2 ratio<−0.58 (p<0.05) and are expected to be either early AR genes (because this is the only differentiated cell type present in fertile, and it is absent in msca1), or just early anther genes that are turned off in msca1. Some genes just above the cutoff are listed because of their importance to either alternative metabolism or ROS handling. Included in this list are glutathione S-transferases, genes involved in alternative metabolism (including 2 genes from the glyoxylate cycle), and a number of hormone biosynthesis genes, most notably, the lipoxygenase protein mutated in the tassel seed1 loss of function mutant, in which no jasmonic acid is produced and the result is femininization of the tassel. Collectively, these data indicate that MSCA1-dependent specification of AR cells activates genes that are responsible for sex determination, ROS management, and organ identity.
Multicellular sexual life cycles initiate with the dedication of cells to a meiotic fate. Such germinal cells commonly conduct several mitoses preceding meiotic entry, however relatively little is known about what characterizes meiotic commitment in these initially fated cells. To redress this, a precisely staged cohort of germinal cells was isolated from maize anthers and compared to somatic niche layers, just 36 hours following their shared derivation from somatic stem cells and six days prior to meiosis. Microarray hybridization provided the earliest transcriptomes of such cell types for any organism: 2529 germinal and 4551 somatic transcripts were either specific (ON/OFF) or differentially regulated (UP/DOWN). There is strong support for the concept that plant germinal cells are hypoxic and curtail reactive oxygen through alternative energy-generating pathways, circumventing mitochondrial respiration. The pre-meiotic set included 116 genes previously classified as meiosis-specific, along with ribosomal components, RNA helicases, pumilio translational repressors and other genes involved in post-transcriptional gene regulation. Additionally, three novel ARGONAUTE genes putatively involved in genome surveillance or chromatin remodeling characterized the germinal cells. These findings establish new properties of just-specified germinal cells, including precocious expression of meiosis-associated functions, and implicate new roles for transcriptional and translational control defining the commitment to meiosis in plants.
A fundamental difference between the kingdoms is the existence of a germ-line in animals and its absence in plants. In most animal phyla, germ-line stem cells are sequestered during early embryogenesis and dedicated to continuous gamete production in adulthood. Male reproductive organs resemble an assembly line, with diploid germ-line stem cells at one end, haploid gametes at the other, and a continuous developmental gradient in between. In contrast, most plants and fungi produce cohorts of germinal cells late in life from a pool of somatic stem cells. In anthers, the male reproductive organ of seed plants, pluripotent floral stem cells rapidly acquire a germinal or somatic fate, exhausting the entire stem cell reservoir. In maize this process yields about 12 archesporial (AR) initials per anther lobe. These enlarge and cycle through 3-4 mitoses before synchronously initiating meiosis; the somatic cells form three layers that provide nutritional and structural support (
Transcriptome profiling has illuminated aspects of anther development at pre-meiotic, meiotic, and post-meiotic stages in rice, maize, and Arabidopsis. Mixed populations of meiotic cells and male gametophytes have been profiled, however, no studies have analyzed isolated, just-committed, pre-meiotic cells, reflecting their inaccessibility within flowers. By exploiting large anther size, reliable staging, and a bulls-eye organ structure that facilitates laser-capture microdissection (LCM) (
Maize AR cell expansion signifying fate acquisition initiates in late day one anthers, but the only AR marker, Sporocyteless of Arabidopsis, is not expressed until the equivalent of maize day five. To identify genes critical to reproductive fate acquisition, the LCM-collected samples were first contrasted with anther primordia, which contain the L2-d stem cells in addition to presumptive vasculature and epidermis (
Filtering for high enrichment and abundance (log fold change>2, expression>median) we identified 49 (3 newly expressed) germinal and 244 (16 new) somatic transcripts as promising cell- and stage-specific markers. A subset was selected for validation of cell-type specificity by qRT-PCR ( 52/59 confirmed) (Table 8) and RNA in situ hybridization ( 15/17 confirmed, 2 gave no signal). Six probes that hybridized to AR cells are the first monocot and earliest plant germinal cell markers reported (
We demonstrate that germinal cells utilize multiple routes for generating ATP and reducing power without respiration. Germinal cells were significantly enriched in phosphoenolpyruvate (PEP) carboxylase kinase, which is regulated by hypoxia and phosphorylates PEP carboxylase, also AR-enriched, to activate cytosolic ATP production. The germinal set also included pyruvate dehydrogenase kinase2 diverting pyruvate away from the citric acid cycle (TCA) towards other AR-specific or -enriched enzymes that convert it to ethanol (pyruvate decarboxylase and alcohol dehydrogenase) or lactate (malate dehydrogenase) and regenerate NAD+ (
Having established morphological and now molecular and metabolic properties of pre-meiotic cells, we asked whether these cells were preparing for meiosis. AR cells were specifically enriched for 34.3% ( 102/297) of genes assigned to maize anther meiotic progression, along with 14 others with defined roles in meiosis (Table 9). These included genes responsible for chromosomal pairing, synapsis, and recombination, including DYAD/SWI1, AFD1, PHS1, homologs of RAD51 and ZYP1, and nine transcripts for DNA repair or double stranded break formation. Therefore, meiotic factors are synthesized just following germinal specification, ˜3 mitoses prior to canonical “pre-meiosis”, challenging current dogma that the meiosis decision point is after pre-meiotic S phase. Precocious expression may permit gradual dilution of mitotic chromatin components during the AR transit amplifying divisions, a hypothesis gaining support for the animal germ-line.
An alternative explanation is that some mRNAs encoding meiotic proteins are stored pre-meiotically, perhaps in the AR cells' conspicuous nucleoli, known sites of ribonucleoprotein (RNP) complex biogenesis and function. RNP-based mRNA protection and storage is a well-established aspect of animal germ-lines. While RNPs have been described in plants, reproductive roles remain undefined. Our data indicate that plant germinal cells express numerous transcripts encoding RNP complex components (6 germinal versus 0 somatic), RNA helicases (14 versus 2), PUF/Pumilio translational repressors (5 versus 0), ribosomal proteins (97 versus 14), and translation initiation or elongation factors (18 versus 6). Collectively these transcripts account for 16.3% of AR cell GO terms, compared to 3.2% of somatic cell GO terms. The abundance of ribosomal components suggests that germinal cells are acquiring the ability to boost translational capacity or build functionally distinct ribosomes. These findings may explain how maize meiocytes constitute just 1.5% of anther cells but contain 20% of anther RNA, much of which will contribute to haploid cell cytoplasm (either as stored RNA or translated protein) following meiosis. Although the mechanism(s) underlying germinal fate specification are widely divergent among animal phyla and plants, RNA-binding proteins are a common feature of pre-meiotic cells.
AR cells also are enriched for numerous genes that affect epigenetic transformations required for reproduction. This is of interest because the timing of de novo DNA methylation, transposable element suppression, and epigenetic reprogramming during the germinal progression is not well understood. Maize has 18 AGO proteins, and we find germinal cells are enriched for five, including AGO105 and AGO121, which cluster with AtAGO4/6/9 involved in RNA-directed DNA methylation. Together with IDN2 and DRD1, genes key to non-CG methylation and also in the AR set. AR cells also precociously express two maize homologs of OsMEL1 (AGO5a and AGO5b) regulating meiotic chromosome condensation. The fifth-most enriched germinal cell marker is the highly expressed AG018a, a strong candidate for interaction with a non-coding class of phased siRNAs (termed phasiRNAs) because of their contemporaneous expression in anthers and specificity to grasses.
For Table 8, the reactions were performed on cDNA made from the same tissue samples as were used for the microarray. qRT-PCR confirmed cell-type specificity for 28/31 germinal transcripts, including two that were classified by qRT-PCR as ON in AR cells and OFF (no amplification) in somatic cells. Six out of nine AR markers tested by in situ hybridization gave the expected AR-localized pattern, one hybridized to both AR and SPL. A further 24/28 somatic transcripts were confirmed, including two that were classified by qRT-PCR as ON in somatic and OFF in AR cells. Eight out of eight somatic markers tested by RNA in situ hybridization gave the expected somatic-specific pattern. In the “validated?” column, a ‘yes’ indicated confirmation of cell-type specificity by qRT-PCR. The requirements for confirmation was that the log 2 ratio of Ct values for the two samples had to be >0.58 in the expected direction. This calculation was made from Ct values that were adjusted for primer efficiency using PCR miner (“www.” followed by “ewindup.info” followed by “/miner/version2/”) and adjusted for starting cDNA amounts by comparison with the housekeeping gene cyanase. All primers were designed to bridge introns and all passed a gDNA and cDNA test with the expected intron size differences between amplified products analyzed by gel electrophoresis. Also indicated on the table are the array log-fold change values for comparison to qRT-PCR results, and the RNA in situ hybridization result, if that experiment was performed for the given transcript.
Table 9. These genes have defined functions in meiosis or were assigned to meiotic progression7 by differential expression in both ameiotic1-pra1 and ameiotic1-489 alleles in meiotic anthers (1.5 mm anther length). The ameiotic1-1 mutant and most other ameiotic1 (am1) alleles in maize have a dramatic phenotype: AR cells look and act normal until meiosis, when they conduct mitosis instead. Am1-pra1 permits meiotic entry but pollen mother cells arrest at the leptotene/zygotene transition, defining the roles of the AMEIOTIC1 protein in two distinct steps of meiosis.
RNA extraction: RNA was extracted with TRIzol reagent (Ambion, Austin, Tex.) from anthers snap frozen on liquid nitrogen within 20 min of dissection, except in the case of laser microdissected cell types (described below). Extraction was followed by chloroform/isopropanol precipitation and resuspension in RNase-free H2O. All RNA samples were DNase treated with RNase-free DNase Set (Qiagen, Venlo, The Netherlands, #79254), purified on a RNAeasy mini spin column (Qiagen, #74104), and quantified on a Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, Mass.).
Laser microdissection: Anthers were fixed in 3:1 ethanol:acetic acid solution, then cryoprotected in 15% sucrose/PBS and embedded in optimal cutting temperature compound (Ted Pella Inc., Redding, Calif. 96069) and frozen, cryosectioned and attached to slides with a Cryojane (Electron Microscopy Sciences, Hatfield, Pa. 19440). After an ethanol to xylenes dehydration series, 10-12 μm sections were laser microdissected using the Zeiss P.A.L.M. Laser Microbeam (“www.” followed by “palm-microlaser.” followed by “com”) for recovery of cell types. RNA was isolated using the PicoPure RNA extraction kit (Arcturus Molecular Devices).
qRT-PCR: mac1 was cloned and the gene encodes the closest maize homolog to TPD1, a putative secreted ligand. We designed primers to the gene for qRT-PCR. We synthesized cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR (kit #18080, Invitrogen, Carlsbad, Calif. 92008) using oligo (dT)20. Each reaction was performed in technical triplicate on cDNA derived from 8-10 ng starting mRNA with SYBRGreenER qPCR SuperMix (Invitrogen, #11762) on an Opticon 2 thermocycler (Bio-Rad, Richmond, Calif. 94547) and fluorescent values were analyzed using PCR miner to account for primer efficiencies. Mac1 transcript was detected using a forward primer in exon 1 (5′-AACCCTACTGCGAAACAACT-3′; SEQ ID NO:1), and a reverse primer that spans exon 2 and 3 (5′-CGAGAATCCTGCGTCCTGAT-3; SEQ ID NO:2) so as to avoid amplifying contaminating genomic DNA. Cyanase was used as a control gene (Forward: 5′-GGTGGTCACATTTGATGGG-3′; SEQ ID NO:3; Reverse: 5′-CTGAGCCCGATACCAACC-3′; SEQ ID NO:4). The ratio of Mac1 to Cyanase was used to normalize expression among biological samples. Each sample type was tested in biological triplicate.
Microarray analysis: For the msca1 and fertile comparison (200 μm anthers), two rounds of RNA amplification and the hybridization were performed as described previously (43). For the laser microdissected AR versus whole anther comparison, two different procedures were used: after RNA extraction, DNase treatment, and RNAeasy column purification, whole anther RNA was quantified and 50 ng of RNA was used for amplification. For AR cell RNA, after laser microdissecction, PicoPure extraction and DNase treatment (described above), RNA was resuspended in water and quantified and 50 ng was used for amplification. Both sample types were amplified according to the Agilent Two-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling Protocol (version 6.5, May 2010) (Santa Clara, Calif.) and hybridized in 4×44K format (Part number: G2519F; Design ID: 016047). Two biological replicates were taken for each sample type for dye swap. Background fluorescence cut off was set and data was normalized. Genes were identified as being above background based on expression at least 3 standard deviations above the mean intensity of negative control probes (false discovery rate p<0.001).
Confocal imaging, and EdU and PI staining were performed as described previously (6). Oxygen probe was an Oxygraph Tx3 (NTH-Pst1-L5-NS40-0.8-YOP) obtained from Presens (Regensburg, Germany). All chemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.) and dissolved in water at the indicated molarities and injected using a 26 gauge needle into the airspace surrounding the immature tassel.
W23 bz2 (deficient in vacuolar anthocyanin accumulation) inbred lines were greenhouse grown in Stanford, Calif. as described previously. Anther length is a reliable indicator of developmental stage1. AR and somatic cells were isolated by LCM and total RNA was extracted from each biological replicate as described previously. We used 0.30-0.35 mm anthers, measured at dissection from the central spike of 2.5 cm tassels. Total RNA was extracted from anther primordia (<0.15 mm), and all samples were amplified and hybridized in 4×44K format (Agilent Part number: G2519F; Design ID: 016047).
Slides were scanned and data were processed. Briefly, the resulting median foreground values for the red and green channels were normalized in two steps using the limma package in R: “within arrays” using the lowess method and “between arrays” using the quartile method. Probes with expression values greater than 3.0 standard deviations above the average foreground of the array's negative controls were considered “ON”, resulting in an estimated false discovery rate of 0.13%. Probes with fewer than 75% of the replicate measurements scored as “ON” were then excluded from further analysis. Significance for differential expression was set at ˜1.5-fold (log 2˜0.58) with a p-value≦0.05. As confirmation, normalized intensities averaged across replicates were compared and presented in supplementary tables as ratios. Anther primordia expression was analyzed in comparisons between <0.15 mm and 0.25 mm and 0.4 mm mac1 mutant anthers.
qRT-PCR was performed as described previously. In situ hybridizations were performed with probe transcribed using the DIG RNA Labeling Kit (T7/SP6). Sense and antisense probes were synthesized from PCR fragments amplified from cDNA clones obtained from the Arizona maize cDNA collection (“http://” followed by “maizecdna.” followed by “org/”). RNA in situ hybridizations were performed on 0.30-0.35 mm anthers residing on the central spike of ˜2.5 cm tassels.
This application claims the benefit of provisional application Ser. No. 61/598,544, filed on Feb. 14, 2012, which application is incorporated by reference herein.
This invention was made with Government support under contract PGRP07-01880 awarded by the National Science Foundation. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/025940 | 2/13/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/123051 | 8/22/2013 | WO | A |
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| Number | Date | Country | |
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| 20140359897 A1 | Dec 2014 | US |
| Number | Date | Country | |
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| 61598544 | Feb 2012 | US |
