Patent Application
20180223301
The present invention relates to a transgenic expression construct for plants, comprising an anti-apoptotic gene of the BAG family of plant genes and a promoter overexpressing said gene in a plant or part thereof. Respective transgenic plants exhibit at least one yield gain phenotype when compared to a non-transgenic plant. The present invention further relates to a method for producing a transgenic plant or part thereof, comprising introducing the transgenic expression construct of the invention into said plant or part thereof, and respective transgenic plants or parts thereof as produced.
Yield in crops and in particular in agricultural crops is a complex multifactorial trait reflecting the interaction of the environment with all growth and development processes. Positive selection over long periods of time has led to a yield increase as a result of genomic changes that impact yield component traits like seed weight and seed number and yield related traits (for example biomass, plant architecture, adaptation and stress resistance).
By 2050, the world population is expected to reach nearly 10 billion. To sustainably provide sufficient food for this increasing population, crop productivity needs to increase by more than 40% compared to the current level. This is a challenge because there is limited potential for future expansion of arable lands whilst at the same time climate predictions suggest that a larger portion of the globe will be subject to erratic environmental conditions.
To date, strategies to increase yield in crops have utilized three major approaches, namely conventional breeding, marker assisted selection and genetic engineering. Of these, genetic engineering promises a great potential since it allows the introduction of selected genes without affecting the desirable characteristics of an elite genotype.
Genetic engineering has been used to improve the resistance of crops against environmental stresses like drought or salinity. Some success has been achieved to reduce the losses caused by these environmental stress factors, which contribute to a major part to crop yield losses worldwide.
While improving the tolerance against environmental stresses may help to reduce the crop yield losses, the solutions found today in this regard do not normally lead to a yield increase compared to the wild type in the absence of such external stress factors.
Programmed cell death (PCD) is essential for all multicellular organisms. The decision of whether a given cell should live or die is the result of a competition between anti-apoptopic and pro-apoptopic (pro-death) proteins and signal transduction pathways.
Compared to the vast molecular knowledge base available for PCD (frequently referred to as apoptosis) in animal or mammalian cells, much less data are available for plant PCD process. It remains undisputed that genetic programs leading to cellular suicide do exist in plants, being involved in resistance to disease and developmental processes. With respect to the latter, PCD pathways are known to play a role, for instance, in the male and female gametophyte development, during incompatible pollen-pistil interactions, during seed development and germination, seed coat and endosperm development, the formation of tracheary elements, aerenchyma formation and lateral and adventitious root emergence, root cap development, leaf abscission processes and senescence.
Plants often respond to pathogen attack by executing PCD programs such as the hypersensitive (HR) response. For instance, plant resistance to biotrophic pathogens are generally a result of the pathogen triggering cell death. Hemi-biotrophs and necrotrophs take over the plant HR response, thus reprogramming plant pathways, tricking the plant to undergo PCD that provides a nutritional benenfit entirely for the pathogen.
Programmed cell death in plants appears distinct from animal apoptosis; plant homologs of mammalian regulators of apoptosis are scarce using standard search algorithms. For example no sequence specific caspase homologs can be found in plant genomes; yet enzyme activities have been demonstrated. Further, there are plant proteases that mediate similar phenomena, with a similar conceptual framework but are not true caspases. Importantly, the ectopic expression of several animal anti-apoptotic genes in transgenic plants is known to confer protection from pathogens and other stresses as described by (Dickman et al., 2001; Lincoln et al., 2002). It is therefore conceivable that the divergent evolution of PCD molecular constituents has led to mechanisms in plants that may be functionally similar to animal genes and pathways independent of primary sequence. However, these cannot be easily deciphered in the absence of searchable sufficiently homologous genes.
Arabidopsis thaliana (At) AtBAG4, Hsp70 and p35 are anti-apoptotic genes that have been reported to confer tolerance to salinity and drought stresses in transgenic tobacco.
The mammalian BAG (Bcl-2-associated athanogene) family of proteins (7 homologs in humans) promote cell survival acting synergistically with Bcl-2. Various additional functions have been ascribed to BAG proteins including apoptosis, tumorigenesis, neuronal differentiation, stress response and cell cycle regulation. BAG proteins are characterized by a common C-terminally localized BAG domain mediating the interaction with Hsp70/Hsc70 molecular chaperones. Additional domains mediate the interaction with the proteasome system, the localization within cells and the interaction with transcription factors (Kabbage and Dickman, 2008).
Heat shock proteins comprise a set of powerful chaperones that are expressed in response to a variety of physiological and environmental stresses and are localized in a variety of sub-cellular compartments. By protein-protein interaction Hsp70 proteins can facilitate anti-apoptotic Bcl-2 proteins to inhibit apoptosis pathways at distinct key points (Joly et al., 2010). The broad-spectrum activity of Hsp70s requires the recruitment of co-chaperones and other chaperone systems (Brodsky and Bracher, 2007). Overexpression of Hsp70 in tobacco conferred tolerance to drought stress (Cho and Hong, 2006).
The function of p35, a Baculovirus anti-apoptotic protein has been extensively studied in different organisms (Clem et al., 1991; Clem and Miller, 1994; Bump et al., 1995; Xue and Horvitz, 1995; Bertin et al., 1996). Expression of p35 in tobacco, tomato and passion fruit significantly improved tolerance to abiotic and biotic stresses (Lincoln et al., 2002; Wang et al., 2009). Transgenic tobacco expressing p35 displayed broad-spectrum tolerance to a range of abiotic stresses including salinity, upon further investigation this tolerance was attributed to the ability to sequester ROS (Wang et al., 2009).
A moderate low-level expression of the AtBAG-4 gene was shown to confer protection to plants from a variety of abiotic stresses, by inhibiting programmed cell death, while knock-out plants were very susceptible to various stresses including drought tolerance, increased salt stress, increased UV-B tolerance, cold treatment and oxidant exposure. In contrast to AtBAG1,5,7, AtBAG6 T-DNA insertion mutants are extremely susceptible to the necrotrophic fungus B. cinerea to which it is normally resistant, suggesting that AtBAG6 has a role in basal resistance limiting necrotrophic pathogen colonization (Kabbage and Dickman, 2008).
Hoang et al. (2015) describe the development of salinity tolerance in rice by constitutive over-expression of genses involved in the regulation of programed cell death. The authors show that the exogenous expression of anti-apoptopic genes from diverse origins, including the AtBAG4 gene of Arabidopsis improves salinity tolerance in rice at the wholeplant level. However, this is related to the avoidance of losses, not to a net yield gain that surpasses standard yield potentials.
In fact, until today no way to improve yield in crops or plants in the absence of environmental stress factors and leading to an improvement in the overall harvest yield compared to the wild-type under the same condition has not been found and still remains a need to be solved.
It is therefore an object of the present invention to provide a process for the increase of yield in crops, in partcilular in the absence of environmental stresses, leading to a yield exceeding the yield in wild-type plants. Other objects of the present invention will become apparent to the person of skill when reading the following specification, examples, claims and figures of the present invention.
According to a first aspect of the present invention, this object is solved by a transgenic expression construct for plants, comprising an anti-apoptotic gene of the BAG family of plant genes comprising at least one BAG domain, or a plant homolog of said BAG gene, and a promoter overexpressing said gene in a plant or part thereof, when compared to the expression the wild-type promoter of said anti-apoptotic gene. Preferred embodiments of the present invention are set forth in the dependent claims and the detailed specification hereinafter.
It was surprisingly found that when an anti-apoptotic gene of the BAG family of plant genes was overexpressed in a plant or part thereof, a substantial increase in yield could be achieved. Their overexpression seems to delay PCD in (e.g.) endosperm cells and has additional effects leading to increased yield potential in monocot (e.g. Oryza sativa) and dicot (e.g. Nicotiana tabacum, Arabidopsis thaliana) plants, upon overexpression. This, for example, affects the grain size for assimilates, but also inflorescence and panicle architecture leading to increased seed numbers. In addition, constitutive expression leads to larger leaves and thicker stems and thus to an increase in overall biomass.
In the context of the present invention, the anti-apoptotic gene of the BAG family of plant genes is characterized in that it comprises at least one BAG domain as described, for example, above. BAG domains are known to functionally mediate the interaction with molecular chaperones, in particular Hsp70/Hsc70. Thus, an “anti-apoptotic gene of the BAG family” shall mean a gene, preferably a plant gene, the product of which a) promotes cell survival (i.e. is anti-apoptotic), and b) comprises a (at least one) BAG domain binding to chaperones, for example Hsp70 and/or 90. The BAG domain can be N- or C-terminally localized. The definition also includes protein complexes, wherein the proteins as involved provide at least one of these “BAG family” functions when present in said complex. Optionally, the BAG-gene can also comprise at least one Ubiquitin-like-domain (see Doukhanina et al., 2006b).
Preferred is the transgenic expression construct according to the present invention, wherein said anti-apoptotic gene is selected from a gene from Arabidopsis thaliana (At)- or Oryza sativa (Os)- or homologs thereof. Seven BAG homologs were identified in Arabidopsis, four of which have domain organization similar to animal members. The remaining three members contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant Bcl-2-associated athanogene (BAG) proteins (Doukhanina et al., 2006).
The following Table 1 shows preferred BAG proteins for use in the present invention.
Other BAG genes that are useful in the present invention are found, for example, in Raphanus raphanistrum, Brassica napus, Brassica oleracea, Raphanus sativus, Brassica rapa, Triticum aestivum, Gossypium hirsutum, Vitis vinifera, Quercus robur, Nicotiana tabacum, Solanum tuberosum, Solanum lycopersicum, Solanum melongena, and Coffea canephora.
A “homolog” of the BAG gene according to the present invention shall mean a gene that encodes for a protein that exhibits at least about 60%, preferably at least about 70%, and more preferred at least about 80% or at least about 90% and even more preferred at least about 95% sequence identity to a BAG-protein and preferably to the/a BAG-domain thereof as described herein, and in particular as above. Of course, this protein also has to be functional with respect to anti-apoptosis and BAG domain, as explained above.
In the context of the present invention, the term “about” shall generally mean a variability of +/−10% of the value as indicated.
In the context of the present invention, a gene is overexpressed by a promoter, particularly in a plant or part thereof, if as a result of said expression an altered spatial distribution and/or an increased quantity of the gene product is found, compared to the expression of the wild-type (regular) promoter for this gene in, for example, a non-transgenic plant or part thereof. Preferred levels of overexpression lead to a more than 2-fold, more than 5-fold, more than 10-fold or even more than 20-fold higher expression of the gene, when compared to the expression of the wild-type (regular) promoter for this gene. Overexpression can be measured using well-known methdos in the art, such as mRNA detection (e.g. using rtPCR), promoter activity assays, and Western blotting (immunoblot) analysis.
In the context of the present invention, it is important to distinguish yield potential and potential yield. Yield potential is defined as the yield of a cultivar when grown in environments to which it is adapted, with nutrients and water non-limiting and with pests, diseases, weeds, lodging, and other stresses effectively controlled. As such, it is distinguished from potential yield, which is defined as the maximum yield which could be reached by a crop in given environments, as determined, for example, by simulation models with plausible physiological and agronomic assumptions (see, for example, Evans, L. T., and R. A. Fischer. 1999. Yield Potential: Its Definition, Measurement, and Significance. Crop Sci. 39:1544-1551).
Hoang et al. (2015) describe that exogenous expression of anti-apoptotic genes from diverse origins, AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improved salinity tolerance in rice at the whole plant level. They demonstrated traits associated with stress-tolerant transgenic events including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Anti-apoptotic genes are said to facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival under salt stress. AtBAG4 from A. thaliana, Hsp70 from Citrus tristeza virus and p35 from Baculovirus driven by the maize polyubiquitin-1 (Ubi-1) promoter were cloned into the binary vector pCAMBIA1301.
Hoang et al. (2015) disclose a yield gain only in the well-known context of protection from salinity stress. Under regular conditions, no improved effect was found when expression the gene (see suppl. data, in particular
For the transgenic expression construct of the present invention, any suitable ubiquitous or tissue-specific plant promoter can be used, as long as the promoter causes an overexpression of the gene. Respective promoters are known to the person of skill, and described in the state of the art (e.g. Dutt et al., 2014). Preferred is a transgenic expression construct according to the present invention, wherein said promoter overexpressing said gene is selected from ubiquitous and tissue-specific plant promoters, such as BSV, CaMV 35S, TA29, actin, FBP7, CMPS, Lhca3.St.1, Mannopin synthase, RolD, Uep1, Ubiquitin and NapA, Gtl (synonymously termed GluA-2), 2S, CuMFT1, Dc3, HaG3-A, LeB4, LegA, Phas, Psl, Str, and USP. For instance, to express a gene only in grains and not in leaves, a seed-specific promoter can be used. The codons of the gene can be optimized for the organism in order to overcome codon usage bias.
Yet another aspect of the present invention then relates to a method for producing a transgenic plant or part thereof, comprising introducing the transgenic expression construct according to the present invention as described above into said plant or part thereof.
Methods for making transgenic plants or parts thereof are known to the person of skill, and may involve, for example, transfer with Agrobacterium tumefaciens, the “gene gun” technology, electroporation or microinjection.
Consequently, another aspect of the present invention relates to a transgenic plant or part thereof, produced according to the method according to the present invention. Said transgenic plant or part thereof according to the present invention, preferably overexpresses an anti-apoptotic gene of the BAG family of plant genes comprising at least one BAG domain as described above, or a homolog of said BAG gene, such as, for example AtBAG4 or OsBAG4 as described above. Most preferred are transgenic plants comprising Gt-1 promoter-driven At-BAG4 or OsBAG4.
In general, the transgenic plant or part thereof according to the present invention can be any monocot or dicot plant, and in particular is a plant used in agriculture, e.g. a crop plant. Thus, preferably the transgenic plant is selected from Arabidopsis, and crop plants, such as wheat, rice, maize, barley, rye, sorghum, soy, sugar cane, pumpkin, tobacco, coffee, wine, broccoli, cauliflower, cassava, potato, and cotton.
Further preferred is the transgenic plant or part thereof according to the present invention, wherein said overexpression is non-ubiquitous (i.e. not in every part or tissue of the plant, or in substantially all parts or tissues of the plant), such as, for example, tissue-specific, e.g. specific for meristematic tissues, fast growing tissues-specific, endosperm-specific, leaf-, root-, stem-, flower-, and/or fruit-specific, or specific for certain plant cell types.
It was surprisingly found that the transgenic plants or parts thereof according to the present invention exhibit at least one yield gain phenotype, when compared to a non-transgenic plant, preferably a plant of the same variety or species without the transgene, or even without any transgene. A yield gain phenotype in the context of the present invention shall mean an increase of the mass per plant, when compared to a non-transgenic plant, also leading to an increase of mass, or grains or seeds, generated from a unit of land expressed as kilograms per hectare. According to the invention, sub-aspects of the yield gain can be selected from larger and/or longer ears or panicles, a tissue specific yield gain, a yield gain under no stress conditions, larger seeds with increased weight, seeds having an increased number of endosperm cells, and larger seed hulls having an increased number of cells.
It was furthermore found that, when employing the present invention, substantial yield gains, e.g. as defined above, could be achieved in the glasshouse. In general, yield gains of a few percentages already result in quite substantial gains. A yield gain according to the present invention is selected from more than about 5% increase in seed mass, more than about 5% increased seed weight per plant, more than about 5% increased seed number, and more than about 5% increase in overall plant biomass such as, for example, more than about 5% of biomass in rice or tobacco plants in the field. In some instances, a yield gain can be about 5%-10% increase in seed mass per plant, and about 5%-15%, preferably about 10%, increase in overall plant biomass, or even more. As can be seen in the examples below, a grain yield gain per plant of up to 60% (rice) or a yield in overall biomass (dry mass) of up to 43% (tobacco) as found in the glasshouse can be achieved.
In more detail, preferred transgenic constructs, such as AtBag4 overexpression in rice using the Gt1 promoter (endosperm- and meristematic- and fast growing tissues-specific promoter), showed 10% longer seeds (due to increased endosperm cell number, longitudinal), and 10% higher mass. The additional yield parameters such as increased panicle length accommodating an increased number of seeds per plant contribute substantially to the strongly increased grain yield noted in
Yet another aspect of the present invention then relates to a method for identifying a yield gain plant or part thereof, comprising identifying the overexpression and/or a modification of an anti-apoptotic gene as described herein selected from genes of the BAG family of plant genes comprising at least one BAG domain, or a plant homolog of said BAG gene in said plant or part thereof, compared to a plant not overexpressing said anti-apoptotic gene and/or comprising said modification, and identifying said yield gain plant or part thereof based on said overexpression. Preferred is the method according to the present invention which further comprises identifying the underlying marker for said overexpression yield gain and/or identifying said modification, and preferably further isolating said marker.
This aspect of the present invention allows to select for and identify yet unknown favorable alleles (mutants) of genes of the BAG family of plant genes comprising at least one BAG domain, or a plant homolog of said BAG gene(s) that lead to a yield gain phenotype as described herein. The yield gain preferably is the result of an overexpression of said gene, but can also be based on functionally improved modifications, such as mutations or gene duplications of said BAG gene(s). The plants used for this method can either be obtained from spontaneously mutated plants, but the method can also comprise a mutagenesis step (for example using gene editing, such as, for example introduction of mutations by Crispr-Cas or TALEN), and then a screening for respective plants and/or markers. Consequently, another aspect of the present invention relates to a plant or part thereof as identified according to the method according to the present invention.
As above, a yield gain phenotype in the context of the present invention shall mean an increase of the mass of a plant or parts thereof, when compared to a non-transgenic plant, including but not restricted to mass of grains or seeds, leaves, stems, roots, tubers and fruit generated from a unit of land expressed as kilograms per hectare. According to the invention, sub-aspects of the yield gain can be selected from larger and/or longer ears or panicles, a tissue specific yield gain, a yield gain under no stress conditions, larger seeds with increased weight, seeds having an increased number of endosperm cells, and larger seed hulls having an increased number of cells. As above, a yield gain according to the present invention is selected from more than about 5% increase in seed mass, more than about 5% increased seed weight per plant, more than about 5% increased seed number, and more than about 5% increase in overall plant biomass such as, for example, more than about 5% of biomass in rice or tobacco plants in the field. In some instances, a yield gain can be about 5%-10% increase in seed mass per plant, and about 5%-15%, preferably about 10%, increase in overall plant biomass, or even more.
The inventors studied PCD-related processes occurring in the developing rice grain and during panicle development as a pathway that has the potential of increasing yield, when down-regulated. They showed that a player of canonical PCD, the anti-apoptotic AtBAG4 gene, delays PCD in endosperm cells and has additional effects leading to increased yield potential in monocot (Oryza sativa) and dicot (Nicotiana tabacum, Arabidopsis thaliana) plants, upon overexpression. This affects the grain sink size for assimilates, but also inflorescence and panicle architecture leading to increased seed numbers.
Further advantages, properties, and features of the present invention become apparent by the following description of preferred examples of the present invention with reference to the enclosed Figures, nevertheless, without being limited thereto. For the purposes of the invention, all references as cited herein are incorporated by reference in their entireties.
SEQ ID No. 1 to SEQ ID No. 8 show amino acid sequences of preferred BAG-genes (see table 1).
Abbreviations: CTAB, Hexadecyl trimethyl-ammonium bromide; DAF, days after flowering; DAPI, 4′,6-Diamidino-2-phenylindole; FDA, Fluorescein diacetate; JC-1,5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethylbenzimidazol-carbocyanine iodide; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
Material and Methods
Chemicals Used
Phusion™ High-Fidelity DNA Polymerase was a product of Finnzymes. Nylon membranes (positively charged), DIG High Prime DNA Labeling and Detection Starter Kit II were from Roche. The following chemicals were purchased from Sigma-Aldrich: JC-1, DAPI, FDA, chemicals for Gibson cloning, cellulose from Trichoderma viride, alpha-amylase from Bacillus licheniformis, Feulgen reagent and toluidine blue.
DNA Constructs
HvSOD (Database Acc. No. AK248365.1) lacking its predicted 40-amino-acid transit peptide (ChloroP 1.1 software), individually or in the combination with a 5′-fusion transit peptide of Pea ribulose-1,5-bisphospate carboxylase/oxygenase small subunit (SSU), were synthesized (GenScript), respectively. Both were equipped with a 3′-BamHI and a 5′ HindIII site. The excised fragments were ligated into an accordingly digested pCambia1380 -derived binary vector harboring the hptll selectable marker gene and the 1.8 kb rice endosperm-specific glutelin Gt1 (syn. GluA-2) promoter (Okita et al., 1989; Wu et al., 1998). This yielded pC1380-Gt1-SSU-SOD and pC1380-Gt1-SOD.
AtBAG4 was amplified using Phusion™ High-Fidelity DNA Polymerase and suitable primers. The purified, Small SmaI/SalI-digested PCR product was inserted into the same binary vector. The OsBAG4 fragment was obtained by BamHI digestion from the original vector (pYC30-OsBAG4) and ligated into the BamHI digested and dephosphorated backbone of the same binary vector. The resulting vectors, pC1380-Gt1-AtBAG4 and pC1380-Gt1-OsBAG4, were used for Agrobacterium transformation.
To determine the tissue-specificity of the 1.85 kb Gt1 promoter used, the vectors pCA1380-Gt1-NLS-3vYFP and pCA1380-Gt1-NLS-GUS were constructed using the same binary vector. The source vectors containing NLS-3vYFP (pMU006) and NLS-GUS (pNK186) were kindly provided by T. Laux (University of Freiburg). The NLS-3vYFP fragment was obtained by BamHI/BglII digestion and ligated into the BamHI digested and dephosphorated backbone of the same binary vector. The NLS-GUS fragment was obtained by PCR amplification using pNK186 as template and suitable primers. The PCR product was mixed with BamHI/SpeI digested backbone fragment and the ligation was done by Gibson cloning (Gibson et al., 2009). All vectors were verified by sequencing.
Rice Transformation and Plant Propagation
Embryogenic calli derived from immature embryos of an experimental Golden Rice 2 (GR 2 (Paine et al., 2005; Ye et al., 2000) event in an Oryza sativa var. japonica (cv Kaybonnet) background (hereafter termed “control”) were used for the co-cultivation with Agrobacteria, hygromycin selection and plant regeneration as described (Ilg et al., 2010). After 3-4 rounds of hygromycin selection, resistant calli were subjected to regeneration. Plantlets were transferred to rooting medium for 2 weeks, and then to Yoshida solution (Yoshida et al., 1976) for 2 weeks at 28° C. under 16 h/8 h light-dark cycle, and transplanted to soil with a spacing of 15×10 cm for greenhouse growth under a 16 h/8 h light/dark cycle at 31° C./26° C. A number of 45-70 independent events were generated for each construct. Preliminary selection for single integration was done by germinating the segregating T1 seed generation in the presence of hygromycin (80 mg l−1). Events with a 3:1 germination ratio were carried into the T2 seed generation, which were subjected to the same selection. Those showing no segregation were assumed to be homozygous. Southern-blot analysis was done to verify the copy number of the insertion. Genomic DNA (20 μg) were digested with EcoRI, a unique restriction site localized adjacent to the 5′ end of the Gt1 promoter. The probes hybridizing with OsBAG4 or AtBAG4 were labeled, and the blotting carried out according to the manufacturer's instructions (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche). The corresponding linearized binary vector plasmids (100 pg/lane) and cDNA fragments generated by PCR or restriction digestion (20 pg/lane) were used as positive controls.
Azygous seeds were isolated from the segregating T1 plant generation using a leaf assay for hygromycin sensitivity, essentially according to (Roy et al., 2012). An optimized concentration of 120 μg ml−1 dissolved in water was applied to 2-3 cm leaf segments of 1-month-old transgenic and control plants and kept in Petri dishes at RT under which condition bleaching of azygous plants became apparent after 5 d. The bleached segments were tracked back to the corresponding plants.
BAG4-Expressing Arabidopsis thaliana and Nicotiana tabacum Plants
The transformation events used expressing AtBAG4 (Acc. At3g51780; NM 115037,7) under constitutive cauliflower mosaic virus 35S (35S CaMV) promoter control (Doukhanina et al., 2006). Tobacco plants (cv Glurk) were transformed with the vector pEarlygate104-AtBag4, resulting in the expression of an N-terminal translational fusion YFP with AtBAG4. The data presented relate to a random mix of four integration events.
Detection of Programed Cell Death
Flowers located at the 3rd to 6th position from the apex of the upper 3 primary branches on the panicle were individually tagged at their flowering date. Freshly harvested seeds at 10, 13, 16, 19 and 22 days after flowering (DAF) from at least 5 plants for each time point were manually dehulled. For the isolation of nuclei, the embryos were excised and the endosperms cut into small pieces, followed by fixation in acetic/ethanol (25:75) over night and then rehydrated with an ethanol/water graded series (75%, 50%, 25%, 0% ethanol). The endosperm samples were kept in a water bath at 60° C. for 10 min and, after rinsing with water, digested over night with 0.5% (w/w) cellulase (from Trichoderma viride (Sigma) and 1250 U ml−1 of Type VII α-Amylase (from Bacillus licheniformis, Sigma) at 35° C. in 1 ml/4 grains of McIlvaine's citrate-phosphate buffer at pH 5.0. The samples were macerated by gently pressing the pellet with a glass rod. After washing with 1× PBS and centrifugation, the pellets were weighted and resuspended in 1× PBS to reach a final concentration of 0.1 g ml−1. A 200 μl aliquot was used for nuclei staining using the Feulgen method as described (Singh and Jenner, 1982). Another 200 μl aliquot was used first for TUNEL staining using the In Situ Cell Death Detection Kit POD (Roche Diagnostics) according to the manufacturer's instruction, followed by centrifugation and resuspension of the pellet in PBS and treatment with a 0.6 μM DAPI solution. This double staining was used to estimate the ratio of viable and apoptotic nuclei. The number of nuclei was determined using a counting chamber with an Axioskop 40 (Zeiss) microscope equipped with corresponding florescence filters. The isolation of nuclei proved to be necessary because TUNEL-detection is not possible in rice endosperm sections (Kobayashi et al., 2013).
The viability of endosperm cells was assessed in 16 DAF seeds with the viability stains fluorescein diacetate (FDA, Sigma-Aldrich) and 5,5′,6,6′-tetrachloro-1,10,3,30-tetraethyl-benzimidazolo carbocyanine iodide (JC-1, Sigma-Aldrich) as described by (Kobayashi et al., 2013). Fresh seeds were dehulled with forceps, and cut into halves submerged in H2O with a razor blade. Sections were treated with 2 mg l−1 FDA in PBS at 25° C. for 25 min in the dark, or 2 mg l−1 JC-1 at 37° C. for 25 min in the dark, followed by 2× washing with PBS at 37° C. Sections of samples were observed and photographed with a fluorescence microscope (Axiovert 135 TV, Zeiss).
Seed Morphology Assessment
For the study of cells in the outer parenchyma layer of the hull, young spikelets section just before flowering were fixed with 2.5% glutaraldehyde in 100 mM potassium phosphate buffer, pH 7.0 and embedded in Epon. Cross-sections of 500 nm were made in the middle part using an Ultra Microtome (Ultracut E; Reichert-Jung) equipped with glass knife and stained with 0.25% toluidine blue. Endosperm cell number was analyzed using cross and longitudinal razor blade hand sections of freshly harvested 22 DAF seeds. Sections were stained with 0.25% toluidine blue and then photographed. Cells were counted using the ImageJ program.
The surface of young spikelets collected just before flowering was visualized by Cryo Scanning Electron microscopy (Cryo-SEM). The samples were mounted on the cryo holder with Tissue Tek O.C.T “glue”, plunge frozen in nitrogen slush, transferred under vacuum into the preparation chamber of the cryo system (Gatan), sputtered with a 20 nm layer of gold and finally transferred into the SEM cold stage. The samples were kept stable at −150° C. under high vacuum. Pictures were taken at 5 kV acceleration voltage. Images were processed using Adobe Photoshop CS3 software. The hull length was estimated with Image J and the cell number in 5th row from the middle side of lemma was counted.
Yield Potential Characteristics
Twenty four siblings of each event were planted in 57×17×10 cm trays (4 siblings per tray) which were randomly placed into the greenhouse and grown under the greenhouse conditions given above. Seeds of homozygous events were first germinated in Yoshida solution for 14 days (Yoshida et al, 1976), and transplanted to soil. The following traits for each plant were recorded at the mature stage: panicle number, seed number per plant, seed setting rate (% of filled grains rel. total spikelet number per panicle), 1000-grain weight and the grain yield in gram per plant. The distribution of seed dimensions was analyzed, after being dried at 37° C. for 3 days using a Microtrac Dynamic Image Analyzer (DIA) and the data evaluated (n=200-2000) with the GraphpadPrism program.
Promoter Characterization
Cellular localization of the histochemical GUS signals and YFP fluorescence was investigated in the T0 generation of transgenic plants. Hand cuttings and inflorescences at progressing developmental stages were collected and photographed. YFP expression in vascular tissues was observed with samples from leaf collar, sheath, cross sections at the 2nd internode, the peduncles of panicle primary branches and the spikelet rachilla. Images were captured with an Axioskop 40 (Zeiss) microscope equipped with the suitable filters for YFP detection.
Results
In brief, the examples show that the present invention led to larger seeds with increased weight in monocot and dicot seeds. Larger seeds are caused by an increased number of endosperm cells, an increased endosperm cell number caused by delayed programmed cell death, an increased number of (larger) seed grains at longer panicles, and larger seed hulls, caused by increased cell number, which allowed the seeds to grow larger. Constitutive expression in tobacco also led to larger leaves, thicker stems caused by more and larger cells and thus, in increased biomass.
Larger Seeds with Increased Weight in Monocot and Dicot Seeds
Larger rice seeds relative to the control were visually apparent in transgenic events in the T2 rice seed generation of transformation events expressing individually the anti-apoptotic OsBAG4 and AtBAG4 gene. This appeared more pronounced with the latter which led the inventors to concentrate on these events. Interestingly, a similar phenotype was observed with events expressing superoxide dismutase from barley. As shown in
A more detailed investigation was conducted with the T5 rice seed generation, after confirmation of single integration by Southern blotting (
In order to assess whether the increased seed size extends to dicotyledonous plants, the inventors examined Nicotiana tabacum (Doukhanina et al., 2006) and Arabidopsis thaliana plants overexpressing AtBAG4 under constitutive promoter control. Arabidopsis seeds were not amenable to 3D analysis because of their too small size, however, they were increased in weight by 24.5% relative to the control (Table 2). Tobacco seeds showed a 30% weight gain (Table 2) which corresponds to an average seed volume gain of ca. 40% and an average increase in length of ca. 10% (
Arabidopsis thaliana
Nicotiana tabacum
Larger Seeds are Caused by an Increased Number of Endosperm Cells
Randomly selected T4 seeds of successful homozygous rice events were harvested and the number of endosperm cells determined in longitudinal and transversal sections. This showed that the number of endosperm cells was increased by maximally 20% in the longitudinal direction (
Increased Endosperm Cell Number Caused by Delayed Programmed Cell Death
Using a DAPI/TUNEL double staining with isolated rice endosperm nuclei, the proportion of apoptotic nuclei containing fragmented DNA was estimated. As shown in
Larger Seed Hulls, Caused by Increased Cell Number, Allow Seeds to Grow Larger
The size and shape of the rice grain is determined by the spikelet hull before fertilization, which consists of the palea and lemma acting as a limiting frame within which grain filling occurs. It is long standing knowledge that, when allowed to grow without this restriction, kernels grow larger (T. Takeda, 1990). Moreover, factors affecting spikelet hull size and shape will impact seed grain dimensions, such as OsSPL16 (GW8), encoding a member of the SBP domain family of transcription factors (Wang et al., 2012), GW2, encoding a RING-type ubiquitin E3 ligase (Song et al., 2007), qSW5, with unknown function (Shomura et al., 2008); OsPPKL1-3, encoding a Kelch-like repeat domain protein phosphatase (Zhang et al., 2012); GS3, encoding a regulatory transmembrane protein and GS5, a putative serine protease (Li et al., 2011).
Given this correlation, the larger seed grains observed upon BAG4 and SOD expression should be preceded by the outgrowth of larger spikelets. As shown in
Gt1 promoter leakiness explains increased spikelet size The increased cell number present in spikelet hulls and the increased hull size are only explicable when strict endosperm specificity of the Gt1-promoter (Okita et al., 1989; Zheng et al., 1993; Wu et al., 1998; Qu et al., 2008) is doubted. The inventors therefore used exactly the same 1.85 kb Gt1 promoter version to drive the expression of the reporter genes NLS-GUS and NLS-YFP in the same (cv. Kaybonnet) rice background. The analysis of the resulting T0 plants revealed promoter leakiness in the developing spikelet's lemna and palea, before panicle emergence (
Preferential expression actively dividing cells is also witnessed by reporter signals in regenerating embryogenic calli and in root tips (
As expected for an endosperm promoter, reporter gene expression was very strong in the endosperm of developing seeds, however, decreased expression is observed at later stages (
Increased Number of (Larger) Seed Grains at Longer Panicles
During early generations of transgenic BAG4 and HvSOD events, the inventors noted that the yield, in terms of gram seeds per plant, exceeded—by far at times—the ca. 10% yield increase that is explicable by increased seed size and weight. This led the inventors to conduct more systematic studies with two selected rice events in the T3 and T4 plant generation (yielding T4 and T5 seeds, respectively) at different times of the year. T4 and T5 seeds were harvested in April and December, respectively, and therefore differed quantitatively in several parameters determined (Table 3). As shown in
Increased Organ Size and Biomass of N. tabacum Plants Constitutively Expressing AtBAG4
Transgenic tobacco plants expressing 35S::AtBAG4-GFP (four events) showed pronounced growth characteristics, in addition to the larger seeds noted above. Young plantlets already showed strongly increased leaf size (
All transgenic plants also showed a pronounced increase in stem thickness (19.4±1.8 mm, diameter; n=7) compared to the wild-type (13.7±2.2 mm, diameter; n=7), thus an average increase of ca. 42%, measured at the 15th leaf position of flowering plants (
At a given age of the plants (shortly before flowering), the leaves were somewhat lower in number in the AtBAG4 overexpressing plants. The latter showed 30.1±1.6 (n=7) leaves while 39±1.6 (n=7) leaves were found in the wild-type (ca. −23%). To cope with this ambiguity po-tentially compensating the increased leaf size, the biomass produced was determined with mature plants (possessing seed capsules) after drying to constant weight at 70° C. (n=7; WT and AtBAG4 overexpressors, each), separated into roots and above-ground parts (seed capsules excluded). This revealed pronounced increases in biomass of the green plant material growing above ground of ca. +43% (Table 5), while the root mass increase of ca. +8% may not be significant.
Taken together, At-BAG4—overexpressing plants show significantly larger leaves (+40%), significantly thicker stems (+42%). They have a reduced leaf number (ca. −23%) at a specific age which does not compensate the increased biomass produced of ca. +43%. This is accompanied by the production of heavier and larger seeds (see above).
Increased Cell Proliferation and Increased Longevity is at the Basis of the Invention
Based on the data presented, our current understanding is that AtBAG4, directly or indirectly, acts as a positive regulator of cell proliferation (the sum of cell division and cell expansion) under standard growth conditions, this forming the basis of the here-presented invention. This is a property that is novel in the field of Programmed Cell Death Research implying that PCD in plants constitutes a continuous process involved in cell homeostasis and morphogenesis. Even in very young leaves (where no PCD is expected), increased cell proliferation is witnessed in the AtBAG4 overexpressing lines (
Increased longevity of plants and of cells is yet another component leading to increased yield potential. Phenotypically, AtBAG4-overexpressing tobacco plants are also characterized by their delayed senescence (
| Number | Date | Country | Kind |
|---|---|---|---|
| 15179384.1 | Jul 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/067707 | 7/26/2016 | WO | 00 |
