Patent Application
20080256664
The present invention relates to means and methods for modifying biomass yield and/or plant growth and/or plant architecture of plants. In particular, it concerns transgenic plants exhibiting an increased biomass yield and plant growth rate compared to the corresponding wild-type plants.
There is a growing interest in the production of plant structures having traits considered desirable by the agricultural or breeding industry. In particular plants wherein the economically useful parts form a large proportion of the modified plants without negatively affecting the health of the plants would be of agricultural, commercial and technical value. To date, there have been only few attempts to modify plant traits, in particular the key trait biomass, by genetic engineering approaches. WO 00/56905 for example discloses a method for promoting or modifying plant growth, yield and architecture in plants comprising the increased expression of at least two cell cycle interacting proteins, more particularly a protein kinase and a cyclin. US 2003/0172402 A1 discloses a method for improving growth, biomass production and xylem fiber length in trees by over-expressing a key regulatory gene in the biosynthesis of the plant hormone gibberellin.
Recently, small RNAs of about 20-24 nucleotides, so-called MicroRNAs (miRNAs), have emerged as important regulators of gene expression. MicroRNAs have been found across both animal and plant kingdoms in great numbers. They have been shown to originate from fold-back RNA precursors, which are processed by the ribonuclease DICER in animals and DICER-LIKE in plants. Plants like Arabidopsis thaliana or animals like zebra fish with mutations in DICER or DICER-LIKE show pleiotropic phenotypes, including lethality for null alleles, suggesting that miRNAs play a very important role in the development of multicellular organisms.
The fold-back structures of plant miRNAs are included in transcripts that are developmentally regulated, may contain introns and be polyadenylated, and are most likely transcribed by RNA polymerase II, thus sharing many features with protein-coding messenger RNAs (Aukerman & Sakai, Plant Cell 15, 2730-2741; Schmid et al, Development 130, 6001-6012).
The mechanisms described for miRNA action require interaction with target transcripts through complementary base pairing, leading to either translational repression or cleavage of the transcript. In animals, miRNAs bind to motives in the 3′UTR of the transcripts. The resulting RNA-RNA interaction, which includes several non-pairing bases, leads to reduced protein accumulation, presumably through attenuation of translation. Compared to animals, target sequences for plant miRNAs are mostly found in the coding regions of the target mRNAs and often share a higher degree of complementary to the miRNAs. Both mechanisms—cleavage and repression of translation—are found to regulate target gene expression in plants. Generally, miRNA targets have turned out to be key regulators of developmental pathways and most of them belong to various classes of transcription factors. As their targets, miRNA precursor genes often belong to families with several loci in the genome that give rise to the same, or very similar, mature miRNA sequences. This mature sequence is in most cases perfectly conserved even in distantly related species, such as between the dicotyledonous plant Arabidopsis thaliana and the monocotyledonous plant rice. Even non-seed plants, such as mosses, share perfectly conserved miRNAs with flowering plants, which demonstrates the general applicability of specific miRNAs to any species of higher plants (Floyd & Bowman, Nature 428, 485-486).
The in vivo functions of most plant miRNAs remain unknown. The microRNA 156 (miR156) of Arabidopsis thaliana was first described in Reinhart et al. (Genes & Development 16, 1616-26) (
The function of SPL genes with predicted miR156 target sequences is also largely unknown, although many were shown to be upregulated upon flowering (Cardon et al; Schmid et al), and one of them SPL3, has been shown to accelerate flowering when overexpressed in transgenic Arabidopsis thaliana plants (Cardon et al, Plant Journal 12, 367-377).
There remains a need to develop a method for modifying plant growth characteristics of plants, in particular traits of technical or economical interest such as biomass yield, plant growth rate and plant architecture. Furthermore, there remains a need for transgenic plants with increased biomass yield, growth rate and modified plant architecture.
Accordingly, the problem underlying the present invention is to provide means and methods which enable the modification of biomass yield and/or plant growth and/or plant architecture and particularly, the provision of genetically modified plants which exhibit a modified biomass yield and/or plant growth and/or plant architecture, in particular an increased biomass yield.
This problem is solved by means of the transgenic plants, seed and propagation materials and the methods as defined in the attached claims.
According to a first embodiment, the present invention pertains to a plant with altered expression of at least one microRNA in the plant compared to a wild-type plant. Altered expression is achieved by any of several means, including selection of knockout lines or naturally occurring variants with altered miRNA expression, modification of upstream regulators of miRNA expression, or transgenic constructs that lead to altered expression of the miRNA or miRNA precursor genes. This plant is characterized by exhibiting a modified biomass yield and/or plant growth and/or plant architecture.
According to a second embodiment, the present invention pertains to a plant with altered expression of at least one microRNA that targets members of the SPL gene family. Altered expression is achieved by any of several means, including selection of knockout lines or naturally occurring variants with altered miRNA expression, modification of upstream regulators of miRNA expression, or transgenic constructs that lead to altered expression of the miRNA or miRNA precursor genes. This plant is characterized by exhibiting a modified biomass yield and/or plant growth and/or plant architecture.
According to a third embodiment, the present invention pertains to a plant with altered expression of at least one microRNA of the miR156 family in the plant compared to a wild-type plant. Altered expression is achieved by any of several means, including selection of knockout lines or naturally occurring variants with altered miR156 expression, modification of upstream regulators of miR156 expression, or transgenic constructs that lead to altered expression of miR156 or miR156 precursor genes. This plant is characterized by exhibiting a modified biomass yield and/or plant growth and/or plant architecture. Part of the invention is further a plant containing altered levels of a microRNA that has at most three mismatches compared to the miR156 family, which targets members of the SPL family of genes encoding SPL transcription factors.
According to a fourth embodiment, the present invention pertains to a transgenic plant that has been genetically modified wherein the genetic modification consists in the introduction of a construct whose presence results in an overexpression of one microRNA of the miR156 family in the plant compared to a wild-type plant. This transgenic plant is characterized by exhibiting a modified biomass yield and/or plant growth and/or plant architecture.
The genetic modification may be any genetic modification leading to an overexpression of microRNAs of the miR156 family in the plant. This encompasses an increased expression as well as a de novo expression of microRNAs of the miR156 family. A corresponding genetic modification may be realized through the introduction of a construct comprising a transgene, said transgene comprising genomic fragments of the miRNA156 sequence being expressed in the plant, thus leading to an overexpression of microRNAs of the miR156 family in the plant. Alternatively, the genetic modification may lead to an overexpression of microRNAs of the miR156 family by use of a modulator acting as an enhancer or a suppressor of gene expression, depending on the circumstances (activation, inactivation).
As used herein, the term ‘construct’ is meant to cover any possible form of genetic information, which provides the information required in the context of the present invention to a particular target cell. The term ‘construct’ includes any nucleic acid such as DNA, RNA, DNA-RNA-hybrids as well as synthetic analogues thereof such as f.i. PNA.
By ‘transgenic’ is meant that a plant according to the present invention comprises at least one construct as defined above stably integrated in the genome.
The sequences of the mature miR156 and various microRNAs of the miR156 family are depicted in
The plants according to the present invention may be of any plant species, including monocotyledonous plants or dicotyledonous plants. Examples of monocotyledonous plants include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats, orchids, tulips and lilies. Examples of dicotyledonous plants include, but are not limited to tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cabbage crops or Brassica oleracea (e.g., cabbage, broccoli, cauliflower, Brussels sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals. Woody species include poplar, pine, sequoia, cedar, and oak.
Preferably, the method according to the present invention is applied to agricultural useful plants cultivated for food or industrial applications. Accordingly, the present invention pertains to fiber producing plants (e.g. flax, hemp, cotton), oil storing plants (e.g. rape, sunflower, soybean), starch storing plants (e.g. wheat, barley, oats, rye, potato, maize, rice, pea, cassaya), sugar storing plants (e.g. sugar beet, sugar-cane) and protein storing plants (e.g. legumes, cereals, soybean). The invention may also be applied to trees, palms and grass (e.g. teff). According to a preferred embodiment, the transgenic plants are plants in which the vegetative parts are of interest (crops grown for silage, lettuce and other green vegetables, etc.).
As used herein, the term ‘modifying plant biomass yield’ means a modified, preferably an increased biomass of either the total plant or of specific parts of the plant such as leaf, shoot, fruit, stem, root, flower, trichome, sepals, hypocotyls, petal, stamen, pollen, style, stigma, seed, embryo, ovule, endosperm, seed coat, nodule, cambium, fiber, aleurone, wood, parenchyma, erenchyma, phloem, sieve element, vascular tissue etc., particularly vegetative parts of plants. Preferably, the biomass of the plant according to the invention is substantially increased compared to wild-type plants by the combined action of significantly higher number of leaves and reduced apical dominance, i.e., an increased number of shoots (branches). Any effect on the biomass yield, compared to the wild type, is part of the present invention. According to preferred embodiments, the biomass yield is increased, favorably substantially increased, and in particular several-fold increased.
As used herein, the term ‘plant architecture’ denotes the morphology of the plants, and relates to various structural features such e.g. the branching pattern, the number, shape, size, texture, position of organs, cells, and tissues of plants, the traits concerning leaves and flower organs being the major determinants for plant architecture. Examples for plant organs and tissues include leaf, shoot, fruit, stem, root, flower, trichome, sepals, hypocotyls, petal, stamen, pollen, style, stigma, seed, embryo, ovule, endosperm, seed coat, nodule, cambium, fiber, aleurone, wood, parenchyma, erenchyma, phloem, sieve element, vascular tissue etc.
According to a preferred embodiment, the plants according to the present invention show the following traits: the leaf number is increased and/or the apical dominance is reduced and/or the number of shoots is increased. The decreased apical dominance may be manifested in that the main shoot is overgrown by side shoots.
As used herein, the term ‘modified plant growth’ relates to an alteration of the growth of the plant, in particular of organs and tissues as mentioned in the foregoing. Preferably, it concerns an increase or acceleration of the growth rate of a plant. According to a preferred embodiment, the increase of the leave production rate is in the range of 1.1 to 1.75.
The present invention is further directed to a method for the modification of the biomass yield and/or plant growth and/or plant architecture of a plant, characterized in that the plant is modified in a way leading to an altered expression of microRNAs compared to a wild-type plant. Preferably, it relates to method for the modification of the biomass yield and/or plant growth and/or plant architecture of a plant characterized in that the plant is transformed with a construct leading to an overexpression of a microRNA of the miR156 family in the transgenic plant compared to a wild-type plant. Regarding further specific embodiments of the methods according to the present invention, reference is made to the above-recited annotations regarding transgenic plants according to the present invention.
A further subject of the present invention is transgenic propagation material, seed material, plant parts and transgenic plant cells. In the present context, propagation materials include any part of a plant suitable for vegetative or sexual propagation for the production of progenies, i.e. for example cuttings, callus cultures, rhizomes, tubers, fruits, seeds, seedlings, protoplast etc.
In the following, the invention will be illustrated by way of non-limiting examples.
In the following examples, miRNAs of the miR156 family were overexpressed in transgenic Arabidopsis plants. These plants produced leaves at a rate that was about 1.5 to 2 times faster than control plants, and they had reduced apical dominance, i.e. an increased number of shoots (branches). These two effects combine to increase the biomass substantially.
Genomic fragments of 1.6 to 2.8 kb in length and centered around the miRNA fold-back sequence (for miRNA156a, b, d and f) as well as a 130 bp genomic fragment containing only the fold-back sequenced (for miRNA156c) were amplified from Arabidopsis thaliana genomic DNA of the ecotype Columbia-0 (see
The genomic fragments were placed behind the 35S promoter of cauliflower mosaic virus (Odell et al) in the pART27 derivative pMLBART (Gleave, Plant Mol. Biol. 20, 1203-1207). The resulting constructs were introduced into Agrobacterium tumefaciens strains ASE (Fraley et al, Biotechnology 3, 629-635). Transgenic Arabidopsis thaliana plants of the reference ecotype Columbia-0 (Col-0) were generated using the floral dip method for Agrobacterium-mediated transformation (Clough & Bent, Plant Journal 16, 735-743).
In the following, the oligonucleotides used for the amplification of the miR156 genomic fragments are depicted.
KpnI, BamHI, SalI denote restriction sites that have been added to the oligonucleotides to subclone the PCR-amplified fragments.
Location of the miRNA genomic fragments that have been overexpressed in plants as annotated by TAIR (www.arabidopsis.org):
156a: chromosome2—nucleotides 10684693-10682418
156b: chromosome4—nucleotides 15074536-15076231
156c: chromosome4—nucleotides 15415545-15415411
156d: chromosomes—nucleotides 3457848-3455606
156f: chromosomes—nucleotides 9134591-9137382
Seeds from plants infiltrated with the miR156 overexpression constructs were stratified at 4° C. for two days, and sown directly on soil. Commercially available Finale® (AgrEvo), which contains 5.78% of ammonium glufosinate, also known as Basta™, was diluted 1:1000, and herbicide-resistant plants were selected by spraying twice a week for three weeks. Plants were grown either in long days (LD—16 hours light, 8 hours dark) or short days (SD—8 hours light, 16 hours dark), at 23° C. and about 150 microEinstein per square meter and second light intensity, provided by a 2:1 mixture of Cool White and GroLux (Sylvania) fluorescent lights.
Similarly to the procedure for Arabidopsis, transgenic Brassica plants (B. napus), tobacco plants (Nicotiana tabacum), and teff grass (Eragrostis tef (E. abyssinica)) were prepared (see
The rosette leaves of transgenic plants according to the present invention were counted at a length of 7 mm. Leaf initiation rate (defined as the number of leaves counted from day X to Y divided by (Y−X) days) was determined. 30-40 plants per genotype were investigated. The results are indicated in Tables 1 and 2.
The results in Tables 1 and 2 show a substantial increase of leaf initiation rate in miR156 overexpressing plants according to the present invention compared to wild-type plants (empty vector).
All leaves of a plant were counted when the plants started to dry out. 10 plants were investigated for each genotype.
The results in Table 3 (and
When the plants started to dry out, all shoots that had formed fruits (siliques) were counted. 10 plants were investigated for each genotype.
The results in Table 4 demonstrate that miR156 overexpressing plants according to the present invention have a higher total number of shoots per plant compared to wild-type plants (empty vector).
In both LD and SD conditions, overexpressing miR156 causes late flowering, which often is accompanied by the formation of aerial rosettes (see
In both LD and SD conditions, plants overexpressing miR156a, b, c and f show decreased apical dominance: the main shoot is overgrown by side shoots. More than 50% of the primary transformants also start to produce flowers first from side shoots rather than the main shoot (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 04008748.8 | Apr 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/03896 | 4/13/2005 | WO | 00 | 6/4/2007 |
